molecular biology of mammalian plasma membrane … · responsible for other aminoacidurias as well...

PHYSIOLOGICAL REVIEWS

Vol. 78, No. 4, October 1998Printed in U.S.A.

Molecular Biology of Mammalian Plasma MembraneAmino Acid Transporters

MANUEL PALACIN, RAUL ESTEVEZ, JOAN BERTRAN, AND ANTONIO ZORZANO

Departament de Bioquımica i Biologia Molecular, Facultat de Biologia, Universitat de Barcelona, and

Departament de Criobiologia i Terapia Cellular, Institut de Recerca Oncologica, Barcelona, Spain

I. Introduction 969A. Amino acid transport systems in the plasma membrane of mammalian cells 970B. Strategies used to identify mammalian amino acid transporters as yet uncloned 973

II. Current Knowledge of the Molecular Structure of Amino Acid Transport Systems 975A. Cationic amino acid transporters 976B. Superfamily of sodium- and chloride-dependent neurotransmitter transporters 985C. Superfamily of sodium-dependent transporters for anionic and zwitterionic amino acids 1003D. Putative subunits of sodium-independent cationic and zwitterionic amino acid transporters 1022

III. Inherited Diseases of Plasma Membrane Amino Acid Transport 1032IV. Prospects 1038

Palacın, Manuel, Raul Estevez, Joan Bertran, and Antonio Zorzano. Molecular Biology of Mammalian PlasmaMembrane Amino Acid Transporters. Physiol. Rev. 78: 969–1054, 1998.—Molecular biology entered the field of mamma-lian amino acid transporters in 1990–1991 with the cloning of the first GABA and cationic amino acid transporters.Since then, cDNA have been isolated for more than 20 mammalian amino acid transporters. All of them belong to fourprotein families. Here we describe the tissue expression, transport characteristics, structure-function relationship, andthe putative physiological roles of these transporters. Wherever possible, the ascription of these transporters to knownamino acid transport systems is suggested. Significant contributions have been made to the molecular biology of aminoacid transport in mammals in the last 3 years, such as the construction of knockouts for the CAT-1 cationic aminoacid transporter and the EAAT2 and EAAT3 glutamate transporters, as well as a growing number of studies aimed toelucidate the structure-function relationship of the amino acid transporter. In addition, the first gene (rBAT) responsiblefor an inherited disease of amino acid transport (cystinuria) has been identified. Identifying the molecular structureof amino acid transport systems of high physiological relevance (e.g., system A, L, N, and x0c ) and of the genesresponsible for other aminoacidurias as well as revealing the key molecular mechanisms of the amino acid transportersare the main challenges of the future in this field.

I. INTRODUCTION the pioneer work of Halvor N. Christensen’s group, differ-ent substrate specificity transport systems for amino acidsin mammalian cells (mainly erythrocytes, hepatocytes,Amino acid transport across the plasma membraneand fibroblasts) were identified (reviewed in Ref. 96), andmediates and regulates the flow of these ionic nutrientsgeneral properties of mammalian amino acid transportinto cells and, therefore, participates in interorgan aminowere revealed: stereospecificity (transport is faster for L-acid nutrition. In addition, for specific amino acids thatstereoisomers) and broad substrate specificity (severalact as neurotransmitters, synaptic modulators, or neuro-amino acids share a transport system). Since these initialtransmitter precursors, transport across the plasma mem-studies, the main functional criteria used to define aminobrane ensures reuptake from the synaptic cleft, mainte-acid transporters have been 1) the type of amino acidnance of a tonic level of their extracellular concentration,(acidic, zwitterionic, or basic as well as other characteris-and supply of precursors in the central nervous systemtics of the side chain) the protein moves across the mem-(for review, see Refs. 93, 96, 97, 505). Transfer of aminobrane, according to substrate specificity and kinetic prop-acids across the hydrophobic domain of the plasma mem-erties, and 2) the thermodynamic properties of the trans-brane is mediated by proteins that recognize, bind, andport (whether the transporter is equilibrative or drivestransport these amino acids from the extracellular me-

dium into the cell, or vice versa. In the early 1960s, after the organic substrate uphill). This functional classification

9690031-9333/98 $15.00 Copyright q 1998 the American Physiological Society

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PALACIN, ESTEVEZ, BERTRAN, AND ZORZANO Volume 78970

has been retained to date, since structural information on ASC, L, y/ and X0AG) and tissue-specific transport systems

(e.g., systems Bo,/, Nm, and bo,/ as well as variants ofhigher eukaryote amino acid transporters is incomplete.Isolation of the first brain GABA transporter (184) in common transport systems). It has been proposed that

this arrangement permits both fine regulation of substrate1990 and the identification of the first cationic amino acidtransporter in 1991 (281, 590) represent the starting points and cell-specific amino acid flows and economy in the

number of structures mediating cellular and interorganfor the cloning of mammalian amino acid transportergenes. Several strategies have been used to identify mam- amino acid fluxes (96, 97).malian amino acid transporters. During the 1980s, severalgroups attempted the purification of these transporters 1. Common systems for zwitterionic amino acidsby different methods. Purification strategies have yieldedfew structural data, although for a couple of transporters The zwitterionic amino acid transport systems A,

ASC, and L are present in almost all cell types. Systemsrelated to neurotransmission (the GABA transporter GAT-1 and the glutamate transporter GLT-1), these data al- A and ASC mediate symport of amino acid with small

side chain with sodium, and system L mediates uniportlowed microsequencing or generation of specific antibod-ies that have been used to isolate cDNA clones (184, 424). of amino acids with bulky side chains (i.e., branched and

aromatic groups). System L has a high exchange propertyAlternative strategies and serendipity allowed the identi-fication of up to 22 cDNA (including splice variants, but (i.e., it shows trans-stimulation, stimulation by substrates

in the trans-compartment) and has been postulated tonot species counterparts) for mammalian amino acidtransporters or related proteins (see sect. II). This struc- serve in many circumstances to mediate efflux from cells

rather than influx into cells (96). Preferred substrates fortural information is not complete. The genes identifiedseem to correspond to eight classic transport systems and system A are alanine, serine, and glutamine, and for sys-

tem ASC alanine, serine, and cysteine (99, 101). In con-their variants, whereas another eight of the major aminoacid transport systems are unknown at the molecular level trast, amino acids with a small, nonbranched side chain

are poor substrates for system L, for which the analogs(see Table 1). An excellent review by Kilberg’s group (342)describes the molecular biology of the amino acid trans- 2-aminoendobicyclo-[2,2,1]-heptane-2-carboxylic acid

(BCH) and 3-aminoendobicyclo-[3,2,1]-octane-3-carbox-porters cloned up to 1995.The molecular identification of amino acid transport- ylic acid (BCO) are model substrates in the absence of

sodium. System L has been subdivided into subtypes L1ers or related proteins leads to ongoing studies on thestructure-function relationship and the molecular genetics (substrate affinity in the micromolar range) and L2 (sub-

strate affinity in the millimolar range), which are presentof the pathology associated with these transporters. Inthis review, attention is paid to the molecular biology, at different ages in hepatocytes and hepatoma cell lines

(597). Therefore, zwitterionic amino acids with bulky sidestructure-function relationship, physiological role, and hu-man genetics of amino acid transporters. Regulation of chains are not cotransported with sodium in many cell

types. This is different in epithelial cells, where broadplasma membrane amino acid transport in mammals isbeyond the scope of the present review and has been specificity systems (e.g., systems Bo, Bo,/) accumulate

these amino acids by coupling the electrochemical gradi-extensively reviewed (96, 278, 350).ent of sodium. One criterion for discrimination of systemA is the fact that it transports N-methylated amino acids

A. Amino Acid Transport Systems in the Plasma (101); in many instances, sodium-dependent transport ofMembrane of Mammalian Cells alanine inhibitable by the model analog N-methylamino-

isobutyric acid (MeAIB) or the sodium-dependent trans-port of MeAIB is used for determining system A transportFunctional studies based on saturability of transport,

substrate specificity, kinetic behavior, mode of energiza- activity. System A is highly pH sensitive. Intracellular his-tidyl residues may form part of the pH sensor of the trans-tion, and mechanisms of regulation performed in perfused

organs, isolated cells, and purified plasma membranes led porter (43, 384). In contrast, system ASC is relatively pHinsensitive. Additional differential characteristics of sys-to the identification of a mutiplicity of transport agencies

in the plasma membrane of mammalian cells (for review, tems A and ASC are that the former is electrogenic andshows the property of trans-inhibition (i.e., inhibition bysee Refs. 26, 94–96). The properties of some of the best-

characterized amino acid transport systems are summa- substrates in the trans-compartment), and the latter maybe electroneutral and shows trans-stimulation, probablyrized in Table 1. From these studies it is evident that a

particular transport system carries different amino acids as a consequence of a high property of exchange. There-fore, whereas system A is considered a true sodium-aminoand that amino acid transport systems show overlapping

specificities. Different cells contain a distinct set of trans- acid cotransporter, system ASC may be an antiporter ofamino acids necessarily associated with the movementport systems in their plasma membranes, as a combina-

tion of common or almost ubiquitous (e.g., systems A, of sodium in both directions (for review, see Ref. 185).


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October 1998 MAMMALIAN PLASMA MEMBRANE AMINO ACID TRANSPORTERS 971

TABLE 1. Best-known amino acid transport systems present in the plasma membrane of mammalian cells

Amino Acid Transport System Isolated cDNA

Zwitterionic amino acids

Sodium dependentA: serves mainly for small amino acids; highly regulated; tolerates an N-methyl group; sensitive to pH changes; trans-

inhibition associated; widespreadASC: excludes N-methylated amino acids; trans-stimulation associated; widespread ASCT1-2N: for Gln, Asn, and His; sensitive to pH changes; so far restricted to hepatocytes; variant Nm in muscleBETA: transports b-Ala, taurine, and GABA; chloride dependent; variants known; widespread Series GAT-1 to -3

BGT-1, TAUTGLY: transports Gly and sarcosine; chloride dependent; variants known; present in several tissues GLYT1-2IMINO: handles proline, hydroxyprolines, and N-methylated glycines; interacts with MeAIB; in intestinal brush-border

membranesPHE: handles primarily Phe and Met; in brush-border membranesBo: broad specificity for most zwitterionic amino acids, including branched aromatic ones; accepts BCH but not MeAIB; ATBo

in brush-border membranes; most probably identical to system NBB (renamed B)Sodium independent

L: mainly for bulky side chain amino acids; trans-stimulated; bicyclic amino acids as model substrates; variantsdescribed; widespread

Cationic amino acids

Sodium dependentBo,/: broad specificity for zwitterionic and dibasic amino acids; accepts BCH but not with MeAIB; in blastocyts,

Xenopus oocytes and probably also in brush-border membranesSodium independent

b/: cation preferring; does not interact with homoserine even in presence of sodium; variants known; in mouseconceptuses

y/: handles cationic and zwitterionic amino acids with sodium; variants known; sensitive to N-ethylmaleimide; CAT-1 to -4widespread

y/L: handles cationic amino acids and zwitterionic amino acids with high affinity only with sodium; insensitive to N- 4F2hcethylmaleimide; in erythrocytes and placenta

bo,/: like Bo,/ but limited by positions of branching; not inhibited by BCH; in blastocysts and in brush-border rBATmembranes

Anionic amino acids

X0AG: similarly reactive with L-Glu and D- and L-Asp; potassium dependent; widespread EAAT1 to -5x0C: cystine competes and exchanges with Glu; in heptocytes and fibroblasts; electroneutral

For anionic amino acids, a capital X is used when sodium dependent. MeAIB, methylaminoisobutyric acid; BCH, z-aminoendobicyclo-[2,2,1]-heptane-2-carboxylic acid.

Transporters ASCT1–2 from the superfamily of sodium- brane. Paradoxically, situations associated with insulindeficiency or insulin resistance (e.g., diabetes, starvation,and potassium-dependent transporters of anionic and

zwitterionic amino acids may represent transporter vari- pregnancy) are associated with upregulation of systemA activity in both hepatocytes and skeletal muscle. Twoants of system ASC (see sect. II). The molecular identity

of systems A and L is unknown. additional long-term maximum velocity (Vmax) regulationprocesses of system A have been demonstrated only inAn important distinguishing feature of system A is

that in many cell types its activity is highly regulated. This culture or incubated cells: adaptative regulation (i.e., re-pression and derepression of system A in the presence orincludes upregulation during cell-cycle progression and

cell growth in many cell types, and hormonal control (in- absence of amino acids) and upregulation by hyperosmo-larity. In both types of regulation, there is indirect evi-sulin, glucagon, catecholamines, glucocorticoids, and

growth factors and mitogens) through a wide variety of dence for the presence of system A regulatory proteins.For a detailed description of the putative mechanismsmechanisms. Thus glucagon and epidermal growth factor

short-term stimulate system A in hepatocytes, whereas involved in the regulation of system A, the reader is di-rected to excellent recent reviews (185, 201, 350, 414) andinsulin upregulates system A in a gene transcription-de-

pendent manner in hepatocytes and through a rapid mech- to specific articles on insulin action in skeletal muscle(188–190, 561). The complexity of the regulation of sys-anism in skeletal muscle. This is independent of protein

synthesis and the electrochemical gradient of sodium, tem A emphasizes the importance of this transport activityin maintaining the accumulation of small side-chain zwit-suggesting direct stimulation of preexisting system A

transporters or recruitment of these to the plasma mem- terionic amino acids as a source for precursor molecules,


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energy, and perhaps even as osmolytes. Unfortunately, tected in kidney and intestine and in other epithelia (192,376, 514). The proline transporter PROT, from the super-the lack of structural information on the system A trans-

porter (see sect. IB1) prevents further understanding of family of sodium- and chloride-dependent transporters ofneurotransmitters, might represent a brain-specific high-the molecular mechanisms underlying system A regula-

tion. affinity [Michaelis constant (Km) in the low micromolarrange] variant of system IMINO; this is not yet clear (seesect. II). The broad-specificity (cationic and zwitterionic2. Tissue-specific systems for zwitterionicamino acids) transport systems Bo,/ and bo,/ are describedamino acidsin section IA3, together with cationic amino acid transportsystems.Additional sodium-dependent zwitterionic transport

systems of restricted distribution have been described(Table 1). Transport of L-glutamine, L-histidine, and L-as- 3. Cationic amino acid transport systemsparagine in hepatocytes has been demonstrated to occurvia a sodium-dependent transport system named N (276). Five transport systems that mediate the uptake of

cationic amino acids are known (Table 1). One corre-A system with similar properties to system N was definedin skeletal muscle and termed Nm (222). System Gly, spe- sponds to the widespread classical system y/, whereas

the other four were discovered in the late 1980s and earlycific for glycine and sarcosine, occurs in several cell types(134). Transporters GLYT1–2, from the superfamily of 1990s (systems y/L, b/, bo,/, and Bo,/) and at present have

been described only in specific tissues. The activity of allsodium- and chloride-dependent transporters of neuro-transmitters, may represent variants of system Gly (see these systems can be distinguished by their affinity for

the cationic amino acids, by their dependence on sodium,sect. II). A transport system for b-alanine, taurine, andGABA (system BETA) has been characterized in several and by their capacity to share transport with zwitterionic

amino acids (for a short review, see Ref. 105). Systemtissues and with differences in substrate affinity and speci-ficity (369). Transporters GAT1–3, BGT-1, and TAUT, y/ catalyzes high-affinity (Km in the micromolar range)

sodium-independent transport of cationic amino acidswhich belong to the superfamily of sodium- and chloride-dependent transporters of neurotransmitters, may be con- and the transport of zwitterionic amino acids with low

affinity (Km in the large millimolar range; affinity increasessidered variants of system BETA (see sect. II).Several zwitterionic transport systems seem to be with chain length) only in the presence of sodium; this

system is electrogenic and accumulates its substrates byspecific to the apical pole of epithelial cells. In intestinalbrush-border membrane vesicles, a sodium-dependent coupling with the cell plasma membrane potential (98,

455, 601). System y/L was discovered in human erythro-transport system (NBB, which stands for neutral brushborder) serving for neutral amino acids is present. This cytes (127) and has also been described in placenta (135,

146). It is possible that system y/L is widely distributedsystem was renamed B for consistency with other broad-specificity systems (e.g., Bo,/, bo,/, b/; see below) (337). among different tissues; thus it has also been detected in

human fibroblasts (D. Torrents and M. Palacın, unpub-More recently, in bovine renal brush-border membranes,a sodium-dependent system for neutral amino acids was lished data). This system transports cationic amino acids

with high affinity (Km in the micromolar range) with nodescribed that was termed Bo (329). Most probably thetransport systems B (NBB) and Bo represent the same need for sodium in the external medium, but it transports

both small and large zwitterionic amino acids with hightransport agency distributed in epithelial cells, as sug-gested after the cloning of the putative transporter for affinity (Km in the micromolar range) in the presence of

external sodium; in the absence of sodium, transport ofsystem Bo (ATBo, from the superfamily of sodium- andpotassium-dependent transporters for anionic and zwit- zwitterionic amino acids is of very low affinity. In addition

to this, system y/ and y/L activities could be discrimi-terionic amino acids; see sect. II). This broad-specificitysystem is thought to be responsible (together with the nated, at least in erythrocytes and placenta, by N-ethylma-

leimide (NEM) treatment, the former being sensitive anddipeptide and tripeptide transport systems; for review,see Refs. 122, 299) for the bulk of renal reabsorption and the latter resistant. Systems y/ and y/L show very high

capacity for trans-stimulation (i.e., exchange). It isintestinal absorption of zwitterionic amino acids. There-fore, system Bo and the ATBo cDNA could represent the thought that exchange via system y/ allows equilibration

of cationic amino acids across the plasma membrane,transport activity and the corresponding transcript thatare altered in Hartnup disease (304), an inherited hyper- whereas heteroexchange between cationic and zwitter-

ionic amino acids plus sodium via system y/L catalyzesaminoaciduria of neutral amino acids (see sect. III). Addi-tional transport systems (see Table 1) seem to be specific the efflux of cationic amino acids against the membrane

potential, the driving force being provided by the sodiumto brush-border membranes. System IMINO, which cata-lyzes sodium-dependent transport of proline and N-meth- ion concentration gradient (14, 90). Systems y/ and y/L

have been suggested to be candidate transport activitiesylated glycines and is inhibited by MeAIB, has been de-


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affected in the inherited disease lysinuric protein intoler- the superfamily of sodium- and potassium-dependenttransporters of anionic and zwitterionic amino acids rep-ance (LPI) (for review, see Ref. 497). This is discussed in

section III. The cDNA for up to five potential y/ transport- resent variants of system X0AG (see sect. II).

Several cell types (e.g., hepatocytes, fibroblasts, anders have been isolated (CAT-1, CAT-2, the splice variantCAT-2a, CAT-3, and possibly the recently identified CAT- embryonic cells) transport L-glutamate (specifically anionic

amino acids with 3 or more carbon atoms in the side chain)4), which form the cationic amino acid transporter family(see sect. II). Several groups described expression of sys- and L-cystine (as tripolar ion) via the sodium-independent

antiport system x0C (28, 533). This system has an apparenttem y/L transport activity in oocytes by the 4F2hc surfaceantigen, which shows homology with another protein, Km in the 100–200 mM range, is insensitive to membrane

potential, and presents trans-stimulation (for review, seerBAT, also related to broad-specificity amino acid trans-port. Because 4F2hc and rBAT are less hydrophobic than Ref. 185). Bannai’s group (27) proposed that this system

participates in a glutamine-cystine cycle that helps cells totypical transporter proteins and form disulfide-bound het-erodimers with unidentified proteins, it has been sug- resist oxidative stress; glutamine, entering the cell via sys-

tems ASC and A, is converted to glutamate, which is ex-gested that 4F2hc may represent a putative subunit ofsystem y/L transporter; this ascription is not yet clear changed for cystine via the oxidative stress-induced system

x0C; accumulated cystine then nourishes glutathione synthe-(see sect. II).Systems Bo,/, bo,/, and b/ were discovered in mouse sis, which protects cells against oxidative insult (27, 29, 596;

for review, see Ref. 228).blastocysts (576–578; for review, see Ref. 573). Amongthe systems that form the series of transport activities forcationic amino acids, the embryonic sodium-independent

B. Strategies Used to Identify Mammalian Aminosystems b/ (subtypes b1/ and b2/, which differ in the

Acid Transporters as Yet Unclonedembryonic stage expression and sensitivity to cationicamino acid inhibition) show the narrowest specificity,

As described in section IA, the present explosion ofserving only for cationic amino acids (576). Systems Bo,/

cloned cDNA related to plasma amino acid transport inand bo,/ show very similar broad specificity with high

mammals is revealing an intriguing range of structuralaffinity (Km in the micromolar range) for cationic and

diversity within amino acid transporter proteins. This di-small and large zwitterionic amino acids. As a distinguish-

versity is already even more pronounced than that showning feature, the former is sodium dependent and inhibit-

by the sodium-dependent glucose transport (for review,able by BCH and BCO, and the latter prefers bulky a,b-

see Ref. 608) and the facilitated glucose transporter iso-unbranched zwitterionic amino acids. More probably,

forms (for review, see Refs. 381, 556). The lack of high-both systems have a wide distribution on epithelial cells.

affinity inhibitors for mammalian amino acid carriers andIn fact, system bo,/ (or a variant, bo,/-like) has been de-

their low abundance in plasma membranes complicatetected in renal epithelial cells and in Caco-2 cells (374,

their structural identification and isolation. Because of557). Expression of rBAT, a protein homologous to the

this, there are many amino acid transport systems not yetcell surface antigen 4F2hc, is needed for system bo,/-like

identified at a molecular level (see Table 1). Among thesetransport activity; it is believed that rBAT acts as a subunit

systems, there are the highly regulated system A and theof this transporter (see sect. II). Mutations in rBAT/system

well-characterized systems L, N, and x0C. In this section,bo,/-like transport activity cause cystinuria type I (for re-

we focus on the strategies used to identify these fourviews, see Refs. 170, 408, 409), an inherited hyperamino-

transporters and also speculate, in some cases, about whyaciduria due to defective renal reabsorption and intestinal

some strategies have failed.absorption of cationic amino acids and cystine (for re-view, see Ref. 487). The role of rBAT in cystinuria is dis-

1. System Acussed in section III.

One of the goals of several laboratories has been toreconstitute and purify system A transporter. Kilberg’s4. Anionic amino acid transport systemsgroup (195) reported the solubilization, reconstitution,and partial purification of system A (70-fold over plasmaL-Glutamate and L-aspartate are accumulated in many

cells (e.g., neurons and glial cells, hepatocytes, entero- membrane vesicles). They then used this protein fractionto immunize mice for the generation of monoclonal anti-cytes, fibroblasts, and placental trophoblasts) by the high-

affinity (Km in the micromolar range) sodium- and potas- bodies. Some of these antibodies specifically coprecipi-tate fodrin and system A transport activity. Because thesium-dependent system X0

AG (165) (for review, see Ref.185). This system shows identical affinity for the D- and protein ankyrin often binds directly to integral membrane

proteins and fodrin, the authors tested whether an anti-L-stereoisomers of aspartate (165). Variants of this trans-port systems occur in neural tissues (100, 147). It is be- body against ankyrin could immunoprecipitate system A

transport activity, and it did. McGivan’s group (437) par-lieved that the five glutamate transporters EAAT1–5 from


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tially purified system A activity from rat liver with conca- the high background (i.e., basal oocyte endogenous activ-ity), and perhaps because the system A transporter wouldnavalin A-affinity chromatography. This demonstrated

that either system A or a protein bound to the carrier is need the expression of different proteins that form partof the whole transporter or are upregulators of its activitya glycoprotein. McCormick and Johnstone (348) purified

system A transport activity from Ehrlich ascites with a (e.g., as shown by the adaptative or osmotic regulation ofsystem A; see above, for review see Ref. 350). In the same30-fold enrichment. Three major peaks were eluted from

the system A-purified Ehrlich cell preparation: high-mo- line of functional expression of system A transport activ-ity, Lin et al. (312) restored normal growth of a mutatedlecular-mass aggregates, a low-molecular-mass band (Ç40

kDa), and the most conspicuous band of 120–130 kDa. yeast cell line incapable of growth in minimal mediumwith proline by transfection with a cDNA (E51) fromInterestingly, polyclonal antibodies against the 120- to

130-kDa purified fraction immunoprecipitated system A mouse Ehrlich cells. This cDNA is 90% homologous to g-actin. Similarly, this cDNA increases sodium-dependenttransport activity. More recently, NH2-terminal sequence

analysis of the 120- to 130-kDa peptide revealed a se- amino acid uptake when expressed in oocytes and in amutated mammalian lymphocyte cell line (GF-17), defi-quence similar to that of the a3-subunit of the a3b1-integrin

(349). Further purification of these extracts using lectin cient in system A transport activity. This suggests that theg-actin-like protein coded for by E51 cDNA may play acolumns resulted in the separation of most of the a3b1-

integrin from the system A activity, indicating that this significant regulatory role in sodium-dependent aminoacid transport. In summary, reconstitution-purificationintegrin is not essential for amino acid transport. More-

over, the fact that transfection of a3-integrin into K562 or and functional expression studies suggest that a multiplic-ity of proteins might be involved in the functional expres-RD cells increased system A transport activity provides

evidence that this protein could modulate this transporter. sion and modulation of system A transport activity.Another approach is the development of cell linesIn summary, from these studies of reconstitution, one may

conclude that several distinct proteins contribute to the that show mutations in amino acid transport activity (re-viewed in Refs. 113, 603). In this way, Englesberg’s groupentire system A transport activity. Probably in the near

future the microsequencing of some of the proteins pres- (371) has isolated constitutive or derepressed mutants forsystem A activity from CHO-K1 cells by alanine-resistantent in these purified extracts will result in the isolation

of all the components necessary for system A function. selection for proline uptake (alar4 mutant) and by a step-wise selection (hydroxyurea treatment and resistance toAnother strategy used to identify system A is the func-

tional expression in Xenopus laevis oocytes. Expression increased alanine concentration) (alar4-H3.9 mutant). Incomparison to the wild type, these mutants showed higherof system A has been claimed (409, 546) based on the

following (for review, see Ref. 277). 1) The effect of gluca- system A activity in isolated plasma membrane vesiclesand higher mRNA-induced sodium-dependent aminoiso-gon on system A in vivo was maintained after mRNA ex-

traction and injection into the oocyte; glucagon is known butyric acid uptake in Xenopus oocytes (371, 546). Thesemutants have increased levels of peptides banding at 62–to stimulate system A transport activity in liver, at least

in part, through mRNA and protein synthesis-dependent 66 kDa and 29 kDa. Sequencing the NH2 terminus of the62- to 66-kDa peptide shows between 80 and 100% identitymechanisms. 2) The apparent substrate affinities reported

by these authors were in the same range as those de- with the mammalian mitochondrial 60-kDa heat-shockprotein (HSP60) (235). Whether these proteins are compo-scribed for the transport of the substrates tested via sys-

tem A. 3) The cis-inhibition of the transport of L-alanine nents of system A carrier is at present unknown.Other approaches involve the chemical modificationinduced by MeAIB suggested that at least part of this

expressed activity was due to system A. 4) The expressed of specific residues by covalent reagents. Hayes and McGi-van (202) identified a 20-kDa protein as a putative compo-transport activity, in contrast to the endogenous uptake,

was inhibited by an extracellular pH of 6.5. 5) Messenger nent of sodium-dependent alanine transport in liverplasma membrane vesicles. On the other hand, the pres-RNA from the Chinese hamster ovary (CHO) cell line

alar-H3.9, which overexpresses system A activity (371, ence of histidine residues critical for activity in the systemA from rat liver (i.e., sensitive to diethyl pyrocarbonate372), resulted in higher transport rates than mRNA from

the parental cell line CHO-K1. More recently, Lin et al. inactivation) has also been demonstrated using this ap-proach (43). Thiol reagents have been used to reveal struc-(313) presented evidence that mRNA of differing sizes (2.2

and 4.2 kb) from two cell lines (GF-14 cells, Ehrlich cells) tural differences between these carriers and between nor-mal and transformed cells. It has been suggested thatincrease the expression of system A transport upon injec-

tion into Xenopus oocytes. However, only the synthesis structural differences occur in system A transporters oftransformed cells, based on the much greater sensitivityof the 2.2-kb transcript is raised by insulin, which is con-

sistent with the idea that there are variants of system A to NEM inactivation of liposome-reconstituted system Aactivity from normal hepatocytes than that from hepa-transporter (see below). Expression cloning of this trans-

port activity has not yet been achieved, possibly due to toma cell lines (132). Moreover, all these studies should


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be critically evaluated, since these specific reagents can high-resolution two-dimensional gel electrophoresis. Twoof these six proteins were microsequenced and show se-modify, with different affinities, a variety of different

amino acid residues in proteins. In any case, no further quence similarity to the mitochondrial heat-shock protein(HSP60). This report and the data published by Engles-structural information on system A has been achieved

with this strategy. berg’s group (235) implicate the family of heat shock pro-teins in the regulation of some transport processes. Howthese proteins develop their function in systems A and L2. System Ltransport activity is unknown.

Using the strategy of functional expression in Xeno-3. System N

pus oocytes, Oxender’s group (521) reported the expres-Kilberg’s group (143), with the same approach of ag-sion of a sodium-independent L-leucine transport system

gregation and differential solubility used to purify systemshared with dibasic amino acids, by injection of mRNAA, achieved a 600-fold enrichment for system N aminofrom CHO cells. This suggests that the expressed trans-acid transport activity in reconstituted proteoliposomesport could correspond to that induced by the expression(540). They identifed a 100-kDa protein involved in systemof rBAT (i.e., system bo,/) and not to system L. In anyN amino acid transport activity by generating monoclonalcase, this ascription is not yet clear, since the data fromantibodies against the purified fraction that immunopre-Oxender’s group (521) and rBAT-expressed amino acidcipitate system N transport activity (539). These tools maytransport activity (549) differ in the sensitivity to inhibi-allow the identification of system N transporter.tion by L-tryptophan. More recently, Broer et al. (68) also

Rennie’s group (551) has reported the expression ofexpressed sodium-independent isoleucine transport activ-rat liver glutamine transporters after injection of rat liverity from mRNA of rat brain in oocytes. The sodium-inde-mRNA into Xenopus oocytes. They attributed part of the L-pendent component of isoleucine transport was inhibitedglutamine-induced transport activity to system N based onby leucine, phenylalanine, and BCH, consistent with thea characteristic feature of this transport system, that is,expression of a system L-like transporter. However, thethe toleration of lithium by sodium substitution and theisolated cDNA responsible for this activity was rat 4F2hc,inhibition by L-histidine in lithium medium. By size-frac-which also expresses cationic amino acid transport intioning the mRNA, they found three different induced trans-oocytes (69). As discussed in section II, several groupsport activities: one sodium independent induced by 2.8–3.6proposed that 4F2hc expresses a system y/L-like trans-kb mRNA, another sodium-dependent, lithium sustitutionport activity in oocytes. In our view, expression of systemintolerant induced by 1.9–2.8 kb mRNA, and one inducedL in oocytes has not been conclusively demonstrated.by a lighter fraction (õ1.9 kb) that is sodium or lithiumAnother interesting strategy to assess the structure ofdependent and that could correspond to system N.this transporter has been developed by Oxender’s group4. System x0c(136). It is based on the fact that the transport activity of

system L can be derepressed by severe ‘‘starvation’’ for Bannai’s group (227), working with manually defolli-leucine or by increasing the temperature of culture in culated oocytes, has reported the expression of a mouse-mutant cell lines with temperature-sensitive leucyl-tRNA macrophage cystine transporter with characteristics ofsynthetase (reviewed in Ref. 603). Oxender’s group (136) system x0c , as it was sodium independent and glutamatetransformed a temperature-sensitive leucyl-tRNA synthe- inhibitable. Fractions of mRNA of 1.5–2.9 kb are responsi-tase mutant CHO cell line (CHO-025C1) with human DNA ble for this induction. Although oocytes seem to expressfrom a cosmid library. Subsequent selection of trans- an endogenous system x0c (574), expression of this systemformants for inability to grow above the permissive tem- correlates with injection of mRNA from x0c -rich cellsperature in the presence of low leucine concentration al- (macrophages stimulated by diethylmaleate in culture),lowed the isolation of cells with higher (õ2-fold) leucine but not from x0c -poor cells (noncultured macrophages anduptake activity. To date, no report has described the res- mouse leukemia L1210 cells). In addition, cystine uptakecue of the human DNA sequences responsible for the expressed by diethylmaleate-stimulated macrophageabove-mentioned transformation. mRNA was, in contrast to the endogenous cystine uptake,

More recently, Segel’s group (606) has developed a pH sensitive, highly temperature sensitive, and inhibitablenew strategy to identify the carrier protein(s) responsible by glutamate. This line of research may lead to the identi-for mammalian L-system amino acid transport. In chronic fication of system x0c transporter.lymphocytic leukemia (CLL), B lymphocytes have mark-

II. CURRENT KNOWLEDGE OF THEedly disminished L-system transport, which is restoredMOLECULAR STRUCTURE OF AMINO ACIDafter treatment with 12-O-tetradecanoylphorbol 13-ace-TRANSPORT SYSTEMState (TPA). These authors identified six candidate L-sys-

tem-related proteins in TPA-treated CLL cells using an L- In this section, the cDNA clones that have been re-lated to plasma membrane amino acid transport in mam-system photoprobe (iodoazidophenylalanine) and ultra-


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mals are discussed from the structural and functional expression in oocytes (106, 108, 242, 448). These cDNA,now named CAT-2 and CAT-2a, represent splice variantspoints of view. With regard to the primary structures eluci-

dated to date, it can be summarized that two main types of (the ‘‘high-affinity’’ or ‘‘T-cell’’ variant CAT-2 and the ‘‘low-affinity’’ or ‘‘liver’’ variant CAT-2a) (see Table 2) of a singlemembrane proteins are involved in amino acid transport:

those that present multiple (i.e., 10–14) transmembrane gene (336). It has been suggested that the three putativeproteins (CAT-1, CAT-2, and CAT-2a variants) may containdomains and are therefore considered as putative trans-

porters and those that do not fit this general model and are 14 transmembrane domains (TM) (9, 106) (Figs. 1 and 2,see below). Very recently, with strategies based on se-considered as activators or as components of oligomeric

transporters. The first group with typical structure of quence homology with CAT-1, the mouse and rat counter-parts (95% amino acid sequence identity between them)transporters is arranged in families of related cDNA: 1)

the family of sodium-independent cationic amino acid of a new brain-specific cationic amino acid transporter,CAT-3, have been isolated (219, 229). Very recently, Sebas-transporters (CAT); 2) superfamily of sodium- and chlo-

ride-dependent neurotransmitter transporters, which in- tio and co-workers (484, 485) identified a human ESTsequence (Table 2) with significant homology to the 5*-cludes amino acid transporters; and 3) the superfamily of

sodium-dependent, and in some cases potassium-depen- end of the open reading frame of CAT-1 and CAT-2/2acDNA (Fig. 1). Screening of the full-length human CAT-4dent, anionic or zwitterionic amino acid transporters. The

second group corresponds to the family of proteins rBAT cDNA [we renamed HCAT-3 (485) as CAT-4 after the re-ported cloning of rat and mouse CAT-3 (219, 229)] hasand 4F2hc that induce sodium- and chloride-independent

amino acid transport with broad specificity (i.e., for diba- been completed, and the putative protein shows 34–38%identity with human CAT-1, -2, and -3 (G. Sebastio, per-sic and zwitterionic amino acids) in X. laevis oocytes. The

members of this family are less hydrophobic than typical sonal communication). At present, there are no reporteddata on the transport activity associated with CAT-4 ex-transporters and show heteroligomeric structure.

Another protein related to amino acid transport, whose pression. Screening, performed by us (DBEST, December1996), for additional expressed sequence tag sequencesprimary structure was elucidated many years ago, is the

anion exchanger band 3 (290). Although its function as an homologous to CAT transporters indicative of additionalmembers of the family was negative.anion exchanger is well accepted, it has been shown to be

associated with the transport of some amino acids (e.g., Figure 1 compares the amino acid sequences of theputative human CAT-1 and CAT-2a proteins, the mouseglycine, taurine, and b-alanine), especially under conditions

of hyposmotic stress; in response to swelling, erythrocytes CAT-3 protein, and the putative NH2-terminal fragment ofthe human CAT-4 transporter. Two potential N-glycosyla-recover their initial volume by releasing organic osmolytes

via a pathway with a pharmacology similar to that of band tion sites, conserved in all known sequences, are locatedin the extracellular loop EL3 in the 14-TM model (Figs. 13 (114, 203, 308). This amino acid transport has the proper-

ties of a volume-sensitive size-limited anion channel (171). and 2). The known amino acid sequences of CAT-2 andCAT-2a (98% identity between the murine counterparts;Interestingly, expression of trout band 3 in oocytes resulted

in anion-exchange activity but also in chloride channel ac- see Fig. 2) are Ç60% identical to that of CAT-1 (106, 448).The rat and mouse CAT-3 show 53–58% amino acid se-tivity and taurine transport (150). At present, it is not known

whether band 3 is involved in the amino acid channel forma- quence identity with the isolated CAT-1, CAT-2, and CAT-2a cDNA (219). Two regions of extensive amino acid se-tion or in its regulation. The role of band 3 in amino acid

transport is outside the scope of this review. This and the quence identity (7E80%) ofÇ150 and 200 amino acid resi-dues, comprising the first three transmembrane domainsmolecular biology of band 3 have recently been reviewed

(10, 380, 574). and transmembrane domains VI–X, are present in theCAT-1, -2, and -3 proteins (334; see Figs. 1 and 2). MurineCAT-2 and CAT-2a differ only in a 41- to 42-amino acid

A. Cationic Amino Acid Transporters segment located in this highly conserved region (intracel-lular loop IL4 between TM domains VIII and IX of the 14-TM model) (Fig. 2). Because a single genomic fragmentFour homologous human and rodent genes defining

a family of cationic amino acid transporters (CAT-1, -2, -3, contains both exons, the isoforms result from mutuallyexclusive alternate splicing of the primary trancript (un-and -4) have been, or are in the process of being, identified

(Table 2). First, expression in oocytes revealed the eco- published data from MacLeod and co-workers quoted inRef. 336). This amino acid sequence region has a role intropic murine leukemia virus receptor (9) (now named

CAT-1) as a putative cationic amino acid transporter (281, substrate binding, as demonstrated by the expression ofCAT-2/CAT-2a chimeric transporters (backbone and the590). Second, full-length cDNA cloned from a previously

identified murine T-lymphoma cell line cDNA (Tea, for 42-amino acid domain) (108).The CAT transporters are homologous to a family‘‘T early activation’’ gene; Ref. 334) showed significant

homology with CAT-1 and cationic amino acid transport of transporters specific for amino acids, polyamines, and


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TABLE 2. Human cationic amino acid transporters

ProteinTransporters (Gene Accession Numbers Human Amino Acid Other Clones in

Name) (Origin of Human Clones) Origin of First Clone and Other Names Chromosome Length Mammals

CAT-1 (SLC7A1) X59155 (Hut78 T-cells) NIH 3T3 fibroblasts (mCAT-1) (9) 13q12-q14 (7) 629 Rat (433, 610)(619) (REC-1, EcoR, ERR, MLV-R

ATRC1, y1/, H13) (105)CAT-2 (SLC7A2) D29990 (intestine) Murine SL12 T-lymphoma cells and 8p21.3-p22 (218) Murine (267)

(218) macrophages (Tea, mCAT-2)murine liver (mCAT-2) (106, 108,242)

Variants: CAT-2(B) (Tea, mCAT-2, 658mCAT-2B and mCAT-2b)

CAT-2A (mCAT-2, mCAT-2a and 657y2/)

CAT-3 (not U70859 (229) (mouse Mouse brain (CAT-3) (229) 619 Rat (219)shown in GDB) brain)

CAT-4 (?) (not EST H05853 (484, 485) 22q11.2 (484, 485) ?shown in GDB)

Accession numbers for human cationic amino acid transporter (CAT)-1, CAT-2, and CAT-2a and for the murine CAT-3 cDNA and one ESTsequence corresponding to human CAT-4 are indicated. Human CAT-4 is tentatively included since the known structure of this cDNA showssignificant homology with other CAT transporters (G. Sebastio, personal communication). GDB, gene data bank; m, murine. Reference numbersare given in parentheses.

choline (APC family) that catalyze solute uniport, solute/ press at least one of the mCAT genes, and in some tissues,cation symport, or solute/solute antiport in yeast, fungi, both genes (CAT-1 and CAT-2) are expressed. Liver is theand eubacteria (448). Marked sequence divergence of only tissue that expresses only mCAT-2a and not mCAT-1these proteins was observed mainly in the hydrophilic or mCAT-2. Kidney, small intestine, resident macrophagesNH2 terminals that precede the first transmembrane heli- and quiescent splenocytes, and T cells only express theces and in the COOH-terminal regions (448). Southern blot mCAT-1 gene but neither of the two mCAT-2 splice vari-studies revealed that all vertebrates examined hybridize to ants. Upon activation, these cell types express the mCAT-the probes of CAT-1 and CAT-2, indicating a high conser- 2 variant. The rat and mouse CAT-3 gene is expressedvation of these proteins among vertebrates (448). Thus specifically in brain, as a transcript of Ç3.4 kb (219, 229).the human CAT-1 protein (7, 619) and the rat CAT-1 pro- The human CAT-4 gene is expressed mainly in pancreas,tein (433, 610) are 86 and 95% identical, respectively, to skeletal muscle, heart, and placenta, and brain, lung, liver,the mouse CAT-1, and the human analogs of CAT-2 and and kidney show a faint band in Northern analysis (Sebas-CAT-2a proteins are Ç90% identical to the murine coun- tio, personal communication).terparts (218; unpublished data quoted in Ref. 105). The tissue and subcellular distribution of CAT trans-

The chromosomal location of the human CAT genes porter isoforms is less known than their transcript distri-is showed in Table 2. The possible involvement of CAT butions. Expression of mCAT-1 protein has been studiedgenes in LPI, a human inherited hyperaminoaciduria that by exploiting its function as a viral receptor (infectivityseems to result from an impairment of a system y/-like or viral glycoprotein gp70 binding assay; Refs. 108, 281,activity (497), is discussed in section III. 590, 610) and by using anti-mCAT-1 antisera (106, 108,

Two excellent recent reviews described functional 605). Recently, approaches to the detection of mCAT-1and structural data as well as available data on the regula- based on its function as a viral receptor have been vali-tion of the expression of CAT-1 and CAT-2/2a transporters dated in null knockout CAT-1 mice (419); primary embryo(105, 336). fibroblasts from these mice were completely resistant to

ecotropic retrovirus infection (i.e., mCAT-1 is the sole1. Tissue expression receptor for ecotropic murine leukemia virus). The lack

of constitutive expression of CAT-1 in human, murine,The tissue distribution of CAT genes has been exam-and rat liver has been demonstrated by virus infectabilityined by Northern blot analysis (for mCAT-1; Refs. 9, 242,studies (610) and immunofluorescence studies (605).281; for hCAT-4, G. Sebastio, personal communication)These strategies served to demonstrate the induction ofand by RT-PCR (specific detection of mCAT-2 and mCAT-surface expression of CAT-1 in the murine liver after par-2a; Refs. 151, 336). Tissue distribution and transcript sizetial hepatectomy and after insulin and dexamethasoneof these genes are indicated in Table 3. Semi-quantitativetreatment (610).data for the murine genes were summarized by MacLeod

and Kakuda (336); all tissues or cell types examined ex- Antibodies directed against the COOH terminus of


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FIG. 1. Amino acid sequence comparison of human cationic amino acid transporters (CAT). HO5853 sequencecorresponds to best EST sequence in database DBEST that showed homology with human CAT-1 and CAT-2, and ratCAT-3, and that served for human CAT-4 cloning (G. Sebastio; personal communication). Amino acid residues presentin all CAT transporter sequences known to date are indicated by gray boxes. Straight lines over sequences indicateputative transmembrane (TM) domains (I-XIV). Between TM V and TM VI, two potential N-glycosylation sites areconserved (open boxes). Dash lines indicate gaps for sequence alignment, which corresponds to that reported by Closs(105).

CAT-2a, which do not distinguish between CAT-2 and sect. IIA5) suggests a high level of regulation of the expres-sion of the corresponding gene promoters and splice vari-CAT-2a, detected a peptide band of 70–80 kDa in liver

(106). Very recently, MacLeod’s and Closs’ groups ob- ants (336). MacLeods’s (151) group has addressed this ques-tion for the mCAT-2 gene. The promoter region of mCAT-tained antibodies capable of distinguishing between CAT-

2 and CAT-2a. This is not an easy task, since the main 2 is extremely complex, with several 5*-untranslated regions(UTR) expanded in a genomic region of 19 kb from the firstdifference between the two mCAT-2 splice variants is an

eight-amino acid segment with five substitutions (see Fig. 5*-coding exon. For more detailed information, the readeris directed to the review by MacLeod and Kakuda (336).2), but full reports on this issue have not yet appeared

(C. MacLeod and E. Closs, personal communication). MacLeod’s lab (151) described five exon 1 isoforms (1a, 1b,1b/1c, 1c, and 1d) that splice into a common sequence 16Therefore, confirmation, at the protein level, of the tissue

distribution and regulation of the expression of the CAT- bp 5* of the AUG start methionine codon (151). Kavanaughet al. (267) described from a liver clone a putative complex2 isoforms is expected to be reported soon. No antibodies

have been reported against the rodent CAT-3 or the human additional 5*-UTR (named 1e) of 515 bp, with six initiationand termination codons that precede the translation startCAT-4 proteins.

The specific tissue distribution and regulation of the codon, which is subjected to posttranscriptional regulation.Promoter 1a (at the far end of the complex promoter region)expression of the different CAT transporter isoforms (see


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FIG. 2. Fourteen TM transmembrane topology model for murine mCAT-2 transporter. Extracellular (EL) andintracellular (IL) loops are numbered. Amino acid residues conserved in all known CAT transporters are indicated ingray circles, and residues that are specific for known CAT-2 and CAT-2a transporters are indicated in white over ablack circle. At bottom, 41- to 42-amino acid segment corresponding to splice variant exon of mCAT-2 and mCAT-2atransporters are compared with homologous mCAT-1 and mCAT-3 regions (residues present in at least 3 of sequencesare contained in gray boxes, and those specific for low-affinity CAT-2a isoform are in white on black boxes). In EL3loop, amino acid sequence that binds viral envelope glycoprotein gp70, present in murine and rat CAT-1, is boxed.Three residues, a Glu in TM III and 2 glycosylated (Y) Asn residues in EL3, are marked by squares, indicating homologousresidues that have been analyzed by site-directed mutagenesis studies of mCAT-1 sequence. Human CAT-1 gene structurehas been published (619), and it has been reported that mouse CAT-1 gene contains 8 coding exons (419). To ourknowledge, gene structure of CAT-2 has not been reported.

predominates over the others in every cell type and tissue in the regenerating rat liver is due to posttranscriptionalregulation, and there is no increased transcriptional activityexamined (336). The promoter of exon 1a is a TATA-less

one with staggered initiation, GC rich, and with several SP1 (run-on experiments) of the gene; this posttranscriptionalregulation is sensitive to cycloheximide. In the rat, CAT-1and CAC boxes. Liver (where only the mCAT-2a variant

is expressed) and activated macrophages (where only the produces two transcripts (7.9 and 3.4 kb in length), whichrepresent alternative polyadenylation signal usage (the longmCAT-2a variant is expressed) use promoter 1a exclusively

(151); this demonstrates that promoter usage does not dic- transcript uses a consensus polyadenylation sequence,whereas the shorter uses a noncanonical signal); both tran-tate the splicing events that render the mCAT-2 and mCAT-

2a splice variants (336). scripts accumulate in the regenerating liver. The specific 3*-UTR of the long transcript contains destabilizing AU-richPosttranscriptional regulation of CAT-1 gene expres-

sion has been reported in liver regeneration (25), a model sequences that are associated with a shorter half-life (90and 250 min for the long and short trancripts, respectively);that induces system y//CAT-1 isoform expression (see sect.

IIA5). This study shows that accumulation of CAT-1 mRNA interestingly, the longer transcript accumulates to higher


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TABLE 3. Tissue distribution and transport characteristics of expressed CAT transporters

CAT Tissue DistributionTransporter (Transcript Size) Expression System Substrates (Km, mM) Cotransported Ligands Trans-Stimulation

CAT-1 Widely expressed (absent Oocytes,e mink L-Arg, L-Lys, L-Orn Na/ dependent, only 8-fold (90, 106, 108)from liver)a (Ç7 and fibroblasts (70–200) for aao(590)Ç9 kb) (588, 589) L-His (Ç2,000) Electrogenic (264,

L-Cys (25) 267)CAT-2 T cell, macrophage, lung, Oocytesf L-Arg, L-Lys (38– Electrogenic (267) 2.9-fold (106, 108, 267)

testisb (Ç4.5 and Ç8.5 380)kb) L-Orn (175–400)

aa7 (low affinity)CAT-2a Liver, muscle, skinc (Ç4.5 Oocytesg L-Arg (2,000–5,000) Na/ dependent, only 1.5-fold (108, 242)

and Ç8.5 kb) L-Lys, L-Orn (NA) for aao(108)Electrogenic (267)

CAT-3 Brain specific (219, 229) COS-7 cells L-Arg (Ç100), L-Lys Yes (229)(Ç3.3 kb) (219), oocytes (Ç150), L-Orn

(229) (NA)CAT-4 Pancreas, skeletal muscle, Not yet reported ? ? ?

heart, placenta úúbrain, lung, liver,kidneyd (Ç2.4 kb)

Transcript size of CAT transporters corresponds to mouse tissues for CAT-1, CAT-2, and CAT-2a, to rat brain for CAT-3, and to human tissuesfor CAT-4. Trans-stimulation is expressed as times that efflux to a trans-side containing 0 substrate is increased when measured at 100 mM trans-side substrate concentration. Apparent Michaelis constant (Km) values were obtained by different labs expressing murine CAT isoforms in Xenopus

oocytes. aao, Zwitterionic amino acids; NA, no data available. References are as follows: a) 9, 107, 242, 281; b) 108, 151, 242, 267, 336; c) 151, 336;d) 106, 484, 485; e) 8, 106, 242, 281, 590; f) 108, 242, 267; g) 106, 108, 268. Other references are given in parentheses.

levels than the shorter transcript. All this suggests that pro- mCAT-2, and mCAT-2a have been studied in Xenopus oo-cytes and are summarized in Table 3.tein factors control the stability of CAT-1 mRNA through

the long-specific 3*-UTR and through common sequences 1) There is sodium-independent transport of cationicamino acids (e.g., L-arginine, L-lysine, and L-ornithine)for both transcripts. In fact, cycloheximide administered in

vivo to control rats upregulates the levels of the long tran- with high affinity (Km in the micromolar range) by mCAT-1 and mCAT-2 (106, 108, 242, 267, 281, 590) and withscript in several tissues but unfortunately not in liver, sug-

gesting tissue-specific regulation of the half-life of CAT-1 low affinity (Km in the millimolar range for L-arginine) bymCAT-2a (106, 267). It is worth mentioning that Km valuestranscripts. Similarly, in rat hepatoma FTO2B cells, where

CAT-1 expression decreases with confluency, the relative for L-ornithine influx are higher than those for L-arginineand L-lysine via mCAT-2, suggesting true differences inabundance of the two transcripts also varies with conflu-

ency; the shorter transcript decreases faster than the longer the extracellular recognition of these substrates (105). Asshown in Table 3, there are discrepancies between labs(610). All this suggests that the 3*-UTR sequences of CAT-

1 transcripts may be involved in the regulation of polyade- as to the Km values of cationic amino acids in mCAT-1and mCAT-2 transporters. Transport of cationic aminonylation and/or stability of the transcripts (25). Unfortu-

nately, in these studies showing differential expression of acids via mCAT-1 is voltage dependent; hyperpolarizationincreases the Vmax and decreases the apparent Km for in-the two CAT-1 transcripts, no attempt was made to correlate

transcript levels and CAT-1 protein abundance to assess flux (the reverse is true for efflux) (264). Closs (105) ar-gued that differences of oocyte membrane potential intranslation efficiency. As an additional regulation mecha-

nism of CAT-1 gene expression, in analogy with CAT-2 gene, different labs and experimental conditions could underliethe variation reported for Km values.these authors quoted unpublished results that suggest multi-

ple promoter usage for the rat CAT-1 gene, but no report 2) For mCAT-1, mCAT-2, and mCAT-2a (242, 267,590), the transport expressed has been shown to be elec-or confirmation of this is yet available.

To our knowledge, information on the promoter regu- trogenic (positive charge follows the cationic amino acidflux) and stereospecific (i.e., Km in the millimolar rangelation of CAT genes to explain the modulation of CAT-

1 and CAT-2 expression (see sect. IIA5) has not been for D-cationic amino acids).3) mCAT-1, mCAT-2, and mCAT-2a present trans-reported.

stimulation of arginine uptake, with mCAT-1 being moresensitive to this phenomenon (106, 108). mCAT-2a trans-2. Transport properties of CAT transportersport activity is largely independent of trans-side substrate(Table 3) (106, 108, 267).The characteristics of the amino acid transport aciti-

vities elicited by the murine CAT transporters mCAT-1, 4) Electrophysiological studies (242, 590) showed


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that mCAT-1 and mCAT-2 transport the zwitterionic to this value (108). Why do mCAT-1 and mCAT-2 not reachthe same accumulation gradient? In the experiments byamino acids homoserine and cysteine only in the presence

of sodium (242, 590). Expression of low-affinity histidine Closs et al. (108), the membrane potential was notclamped, and therefore, the impact of the high L-arginineuptake is also elicited by mCAT-1 and mCAT-2; for mCAT-

1, it has been shown to be partially dependent on the flux (10 mM extracellular concentration) was not con-trolled. Additional work at different extracellular sub-presence of cis-sodium. At low pH, when histidine is pro-

tonated, this amino acid becomes a better substrate, dem- strate concentrations and at constant membrane poten-tials is needed to characterize the accumulation capacityonstrating selectivity of CAT transporters for dibasic

amino acids. For mCAT-2a (106), transport of zwitterionic of these transporters and the transport mechanisms un-derlying any possible difference between them.amino acids in the presence of sodium has not been ob-

served, although in these studies transport activity was For the human CAT-1, -2, and -2a counterparts, as forthe mouse analogs, oocyte expression showed cationicmeasured by radioactive amino acid uptake, a less sensi-

tive method than electrophysiological measurements. amino acid transport of high affinity that was sensitiveto trans-stimulation for hCAT-1 and hCAT-2 and cationicAt present, there are no data available on the amino

acid transport activity expressed by hCAT-4 (but expression amino acid transport of low-affinity that was only slightlysensitive to trans-stimulation for hCAT-2a (unpublishedof hCAT-4 in oocytes resulted in increased L-arginine uptake;

Sebastio, personal communication), and there are two re- data quoted in Ref. 105).On the basis of all these characteristics (transportports on rat and mouse CAT-3 (Table 3). Transient expres-

sion of rCAT-3 in COS-7 cells resulted in sodium- and chlo- properties and tissue distribution), these cDNA (CAT-1,CAT-2, CAT-2a, and CAT-3) have been attributed to sys-ride-independent transport of radiolabeled L-arginine with

an apparent Km of Ç100 mM, inhibitable by cationic amino tem y/ and its variants (242, 281, 590). In summary,transport activity elicited by mCAT-1, mCAT-2, andacids and dependent on membrane potential, as expected

for a cationic amino acid transporter (219). Expression of rCAT-3 expression is similar but with subtle differences(105, 219): sodium-independent high-affinity transportmCAT-3 resulted in high-affinity, sodium-independent trans-

port of dibasic amino acid, which shows trans-stimulation for cationic amino acids, with a slightly higher apparentaffinity for mCAT-1, which is more sensitive to trans-(229). Therefore, CAT-3 together with CAT-1 and CAT-2

transporters are high-affinity cationic amino acid transport- stimulation. No data are reported on trans-stimulationvia CAT-3. The transport properties and tissue distribu-ers in contrast to the CAT-2a isoform.

It is worth mentioning that the proposed channel mode tion of CAT-1, CAT-2, and CAT-3 are consistent withsubtle variants of system y/ (reviewed in Refs. 126, 600),of action described for the sodium-dependent amino acid

transporters, like members of the superfamilies of neuro- the common cationic amino acid transport activity ofmammalian cells. Most probably, CAT-2a represents atransmitters and of excitatory amino acid transporters (see

the corresponding sections), has not been described for the low-affinity liver variant of y/ activity. White and Chris-tensen (601) described a low-affinity transport of L-argi-CAT transporters. It is interesting that these transporters

and the proteins rBAT and 4F2hc, which essentially do not nine in primary hepatocytes not subjected to trans-stim-ulation and concluded that the classical y/ activity wasmediate sodium-dependent transport, do not seem to have

a channel mode of action (90, 110). absent or altered in hepatocytes. However, Van Winkle(574) suggested that the transport activities expressedCloss et al. (108) obtained surprising data on the ac-

cumulation capacity of mCAT-1, -2 and -2a transporters by CAT-1 and CAT-2 could fit those of systems b/. VanWinkle et al. (579) also reassessed mCAT kinetic data,in oocytes at a nonphysiological extracellular concentra-

tion of 10 mM L-arginine. Incubation of oocytes in a high suggesting the presence of both a high- and a low-affinitycomponent for each protein (mainly for mCAT-2a) whenL-arginine concentration (10 mM) for 6 h, assuming an

oocyte space distribution of Ç180 nl/oocyte (90), leads to expressed in oocytes. At present, it is not clear whetherthis complex kinetic behavior represents an artifact of0.6-fold accumulation in mCAT-1-expressing oocytes, 1.4-

fold in mCAT-2-expressing oocytes, and 6-fold in mCAT- the expression model, different conformation or oligo-meric states, or interaction with endogenous proteins.2a-expressing oocytes. These differences have been inter-

preted as the consequence of an apparent intracellular A more careful characterization of these amino acidtransport activities based on inhibition by amino acidssubstrate affinity of mCAT-2a smaller than that of mCAT-

1 and mCAT-2 (105). In our opinion, thermodynamic gradi- and analogs is needed to clarify this issue. Similarly, cellknockout or antisense experiments, like those reportedents are unlikely to be the result of substrate affinity dif-

ferences. The regular oocyte membrane potential (030 to for system bo,/-like in opossum kidney (OK) cells (374),would clarify the contribution of CAT transporters to050 mV) is valid for an accumulation gradient of a positive

charged substrate (i.e., L-arginine) of six- to eightfold. In- y//b/ transport activity in cells. In this sense, uptakestudies in cells derived from the null knockout CAT-1terestingly, accumulation of 10 mM L-arginine in mCAT-

2a-expressing oocytes from a sodium-free medium tends mice (419) may help to clarify this issue.


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not be located extracellularly, which thus favors the 14-3. Protein structure of CAT transporters

TM model.Structural information on CAT transporters is To our knowledge, extensive studies in search of evi-

scarce. All CAT transporters identified lack a characteris- dence for the 14-TM model, like those performed withtic signal peptide, and therefore, the NH2 terminus is con- the GABA transporter GAT1 (38), the glycine transportersidered to be cytoplasmic (see Refs. 105 and 336 for re- GLYT1 (401), and glutamate transporters (585, 498), haveview and Ref. 219 for rCAT-3). Most of the additional not been reported.information available has been obtained from mCAT-1,and it has been extrapolated to CAT-2 and CAT-3 trans-

4. Structure-function relationshipporters since they show almost identical hydrophobicityprofiles (105, 219). These profiles initially suggested two Swapping chimeras with the divergent amino acid seg-membrane topology models for CAT transporters: 12 TM ment of CAT transporters, mutational analysis and studies(according to MacLeod’s and Saier’s groups) or 14 TM related to the interaction with the murine ecotropic leuke-(according to Cunningham’s group) (9, 106, 334, 448). The mia virus provided the core of our knowledge of the struc-two models differ in the middle TM domains (TM domains ture-function relationship for CAT transporters.VII and X of the 14-TM model are considered to be intra- The apparent substrate affinity, maximum transportcellular in the 12-TM model; see Fig. 2). The 12-TM model rate, trans-stimulation, and accumulation capacity are dis-is supported by the fact that CAT transporters belong tinctive features of mCAT-2 and mCAT-2a (see Table 2,to the APC transporter superfamily of yeast, fungi, and CATS). This suggests that these differential transport ca-eubacteria, which presumably contain 12 TM domains pacities are determined by the variant exon coding for(448), whereas the proposed membrane topology of the the 41- to 42-amino acid residue divergent segment of thefirst 8 TM domains of the homologous yeast and fungi two proteins (see Fig. 2). Closs et al. (108) performedpermeases argue in favor of the 14-TM model (105, 508). elegant studies, in which chimeric transporters with theMutational analysis showed that the viral binding site of backbone of mCAT-1 were completed with the divergentmCAT-1 (see Fig. 2) is located between TM V and VI, domain of mCAT-2 and mCAT-2a, and the backbone ofconfirming the extracellular location of extracellular loop mCAT-2 was completed with the corresponding domain(EL) 3 in both models (8). Evidence in favor of the 14-TM of mCAT-1 (see Fig. 2). The transport characteristics (ap-model has been obtained: 1) antibodies directed against parent Km, Vmax, trans-stimulation, and accumulation ofpeptides of the EL3 and EL4 loops of mCAT-1 in the 14- L-arginine) of these chimeras expressed in oocytes areTM model immunostained nonpermeabilized cells (605). similar to those of the divergent region. Interestingly, theThis confirmed the extracellular location of these protein recently cloned rat CAT-3 expresses high-affinity (Km

regions; the 12-TM model predicts an intracellular loca- Ç100 mM) L-arginine uptake in COS-7 cells, and its diver-tion for the EL4 protein region. 2) The glycosylation of gent domain is more similar to that of CAT-1 and CAT-2CAT transporters has been demonstrated by endoglycosi- than to that of CAT-2a (219) (see Fig. 2). These data sug-dase F (endo F) treatment of immunodetected CAT-1 (i.e., gest that the divergent protein domain of CAT transport-antiserum raised against the COOH terminus of murine ers has an impact on all transport properties, and there-CAT-1) and CAT-2/CAT-2a (i.e., antiserum raised against fore, it might have a role in substrate recognition, turnoverCOOH terminus of murine CAT-2a; a region that is identi- number, and the translocation mechanism. As indicatedcal to CAT-2) from mammalian cells or expressed in oo- in Fig. 2, the divergent domain corresponds to the intracel-cytes. These studies showed a broad glycosylated moiety lular loop IL4 (including a few amino acid residues of TMof 3–9 kDa for mCAT-1 and mCAT-2a transporters (106, domains VIII and IX) in the 14-TM domain model of CAT280). Mutation in mCAT-1 of the two putative N-glycosyla- transporters.tion sites Asn-223 and Asn-229 to histidine, conserved in Residues involved in the reported mutational analy-all CAT transporters characterized (Fig. 1), results in a ses of mCAT-1 are indicated in the homologous positionprotein with identical SDS-PAGE mobility to the endo F- in the mCAT-2 protein model depicted in Figure 2. N-treated wild-type mCAT-1; mutation of either Asn residue glycosylation is not required for transport function ofresults in an intermediate mobility. The 12-TM model pre- mCAT-1 (280); the unglycosylated mutant (double Asn todicts an additional, unconserved extracellular N-glycosyl- His mutation at positions 223 and 229: see Fig. 1) ex-ation site in mCAT-1 (Asn-373, located in the intracellular presses an unaffected transport activity in oocytes. TheIL4 of the 14-TM model). Mutation of Asn-373 to histidine glutamate residue at position 107 of mCAT-1 is conserveddoes not affect glycosylation of mCAT-1. These studies in the TM domain III of all known CAT transporter se-(280) demonstrated that Asn-223 and Asn-229 are the gly- quences (see Figs. 1 and 2) and also in the yeast transport-cosylated residues of mCAT-1, and therefore extracellu- ers for arginine, histidine, and choline of the APC familylar, like the loop EL3 in the 14-TM model (Fig. 2), and (589). This residue is required for transport activity in

mCAT-1 protein expressed in mink CCL64 lung fibroblaststhat Asn-373 (in the IL4 loop of the 14-TM model) might


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(589). Substitution by aspartate led to a loss of transport function may be uncoupled, but this is still an openquestion, since conformational changes for the trans-activity; interestingly, substitution by the uncharged gluta-

mine residue did not affect transport activity (data by Kim port-defective mutant have not been ruled out.and Cunningham quoted in Ref. 105). All these substitu-tions led to mCAT-1 protein expressed in the plasma mem- 5. Physiological role of CAT transportersbrane of the transfected cells as demonstrated by its roleas a virus receptor (infectivity and viral glycoprotein gp70 An intriguing question is why there is such a variety

of CAT transporters in mammalian cells. Cationic aminobinding). All this suggests that the carbon backbone sizebut not the negative charge of residue glutamate 107 of acids are needed for protein synthesis, urea synthesis (ar-

ginine), and as precursors of bioactive molecules (argi-mCAT-1 determines transport function for the CAT trans-porters. nine and ornithine are substrates for the synthesis of urea

and nitric oxide as well as polyamines, respectively).Meruelo and co-workers (621) and Cunninnghamand co-workers (8) identified by domain swapping and Then, what does each CAT transporter isoform contribute

to the supply of substrates for these purposes? The contri-mutational analyses the sequence NVKYGE (amino acidresidues 232–237 in mCAT-1) within EL3 (see the corre- bution of other protein structures to cationic amino acid

transport, like rBAT (system bo,/-like) and 4F2hc (systemsponding position of this protein segment in Fig. 2) asessential for virus envelope binding and infection; y/L-like) are discussed in section IID. The nearly ubiqui-

tous CAT-1 isoform most probably corresponds, as dis-swapping the above-mentioned sequence into the hu-man CAT-1 conferred infectivity and virus binding (8). cussed before, to the classical system y/, a high-affinity

and electrogenic cationic amino acid transport systemThis sequence is also present in the rat CAT-1 counter-part (610), but not in hCAT-1 or the known CAT-2 pro- that allows accumulation of these substrates within the

cells for general metabolic purposes. Consistent with this,teins (see Figs. 1 and 2). Interestingly, both mCAT-1 andrat CAT-1 serve as a receptor for the virus. Detailed the null knockout CAT-1 mice are smaller (limited accre-

tion) at birth (419). In this sense, at a physiological extra-description of the amino acid residues within the EL3loop required for binding of the viral protein envelope cellular concentration of L-arginine (50 mM), a high ex-

pression level of mCAT-1 in oocytes allows the mainte-gp70 and permissivity to infection (8, 267) has beenreviewed by Closs (105). It is worth mentioning that nance of an L-arginine gradient across the plasma

membrane of Ç19-fold (90).none of the mutations examined to determine viral in-teraction with CAT transporters affected their transport The known examples of upregulation of CAT-1 trans-

porter expression favor this general role of the classicalactivity. In contrast, interaction of mCAT-1 with thevirus reduces its transport activity. Coexpression of system y//CAT-1 isoform. The expression of this gene is

enhanced in proliferating cells (e.g., T and B lymphocytesmCAT-1 and glycoprotein gp70 resulted in a specificreduction of mCAT-1 glycosylation and transport activ- activated by concanavalin A and bacterial lipopolysaccha-

ride, rapidly growing cells infected with Friend leukemiaity; a decrease in transport activity also occurs with theunglycosylated double Asn to His mutant at residues virus, and a variety of tumor cells of different origin)

(620). In liver regeneration, CAT-1 expression (both223 and 229 (280). Binding of glycoprotein gp70 to thetransfected mCAT-1 results in noncompetitive inhibi- mRNA and protein) is induced a few hours after hepatec-

tomy (25, 610). Recently, Hatzoglou’s group (25) showedtion of amino acid import via the murine CAT-1 with noeffect on amino acid export (588). The effects of gp70 that CAT-1 could be considered as a delayed early growth

response gene in the regenerating liver that requires pro-on transport kinetics led the authors to suggest thatgp70 binding represents a steric hindrance that slows tein synthesis for its upregulation. In keeping with this,

ecotropic retroviruses infect hepatocytes during fetal de-the rate-limiting step of the amino acid import cycle, aconformational transition of the empty transporter in velopment and liver regeneration, but not in adult hepato-

cytes (198). This supports a role of CAT-1 transporter inwhich the binding site moves from the inside back tothe outside of the cell, and that gp70 has no effect on accretion, and as discussed by Wu et al. (610), a role of

this transporter in the supply of ornithine for polyaminethe rate-limiting step of the amino acid export cycle. Asimilar mobile carrier hypothesis has been suggested synthesis. Interestingly, the key enzyme in polyamine syn-

thesis, ornithine decarboxylase, peaks during the G1 phasefor the two-directional operation of the system y/ (600).The above-mentioned data suggest that amino acid (145), and ornithine levels rise after partial hepatectomy

(149). In addition to proliferation, hormone treatment (in-transport and virus receptor functions may be coupled;conformational changes of the transporter may lead to sulin and dexamethasone) also induces mCAT-1 expres-

sion in liver (610). A recent report also links CAT-1 expres-membrane fusion of virus and host cell (105). Interest-ingly, the transport defective mCAT-1 mutant glu107asp sion with cell proliferation. Perkins et al. (419) reported

that the homozygous null knockout CAT-1 mice developmediates binding of glycoprotein gp70 and virus infec-tion (589). This suggests that transport and receptor anemia and die after birth. Erythroid maturation is defec-


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tive in these mice because of a specific defect in cell mechanisms involved in the stimulation of insulin secre-tion by L-arginine in mouse pancreatic b-cells. This workproliferation and/or differentiation. In addition, this sug-

gests that CAT-1 transporter is the main contributor to suggests that L-arginine raises the intracellular concentra-tion of Ca2/ and stimulates insulin secretion as a conse-the cationic amino acid supply to erythroid progenitor

cells. The specific contribution of CAT-1 transporter to quence of its electrogenic transport into this cells. Theexpression of mCAT-2 and mCAT-2a in b-cells was dem-the intracellular accumulation of cationic amino acids in

those cells that express additional CAT isoforms (e.g., in onstrated by RT-PCR. L-Arginine produced a dose-depen-dent increase in the intracellular concentration of cal-brain, heart, skeletal muscle, uterus, ovary, testis, and

placenta) is difficult to assess by indirect determinations, cium, which suggests that the low-affinity mCAT-2a is thecationic amino acid transporter responsible for the secre-like transcripts and protein levels (see below). Specific

knockout and antisense experiments are needed to delin- tagogue action of this amino acid. Specific mCAT-2aknockout experiments in b-cells or in the whole animaleate the contribution of each CAT transporter to the mac-

roscopic amino acid flux through the plasma membrane are needed to demonstrate the role of mCAT-2a in theinsulin secretagogue action of L-arginine.of the cells.

The low-affinity high-capacity transport properties, The CAT-1 mRNA is constitutively expressed in ma-ture resting and activated T cells and splenocytes andthe accumulation capacity through the plasma membrane

at high extracellular substrate concentrations, and the ex- resident macrophages (242, 336). Activation of these cellsmainly induces the CAT-2 transporter isoform. SL12 thy-clusive expression of CAT-2a isoform mRNA, but not CAT-

1 (both protein and mRNA) or CAT-2 (mRNA) isoforms, in moma cell clones, a model system of thymocyte differenti-ation, show developmental regulation of mCAT-2 duringliver have been envisaged as constituting a kinetic barrier

between the hepatic urea cycle and extracellular arginine thymocyte maturation (602). The mCAT-2 gene is down-regulated in normal and mature thymocytes until it is acti-(105). Furthermore, on the basis of the low intracellular

concentration of arginine in liver, it is unlikely that this vated by mitogens or antigens (151, 242, 334). Peripheralblood lymphocytes and quiescent splenocytes exhibit lit-amino acid is released through the activity of CAT-2. All

this is consistent with the lack of activity of the classical tle transport of lysine via systems y/ and y/L (64, 127).Upon activation, T cells rapidly increase system y/ trans-high-affinity system y/ in the hepatocyte plasma mem-

branes, which protects extracellular L-arginine from hy- port activity and mCAT-2 mRNA levels in parallel (64). Inaddition, a transient increase in mCAT-1 expression hasdrolysis by hepatic arginase (600, 601). The hepatocyte

CAT-2a transporter would allow rapid accumulation of been reported (336). Human CAT-1 antisense experimentsin phytohemagglutinin-induced lymphocytes reduce thecationic amino acids only at high plasma concentrations,

leaving sufficient substrates in circulation for cells ex- induced system y/ transport activity only partially (88).Combined experiments with antisense sequences of CAT-pressing the high-affinity CAT isoforms (105). In keeping

with this, expression of CAT-1 isoform occurs in liver 2 and CAT-1 have not been addressed.The release of nitric oxide is an important mediatorwhen the urea-cycle enzymes are downregulated (e.g.,

liver regeneration, insulin treatment, low-protein diet) of macrophage function (389, 536). Activation of macro-phages by bacterial lipopolysaccharide (LPS) and inter-(610). Interestingly, stress conditions (partial hepatec-

tomy, surgical trauma, and fasting) upregulate mCAT-2a feron-g (IFN-g) produces the parallel induction of theexpression of CAT-2 (108) and the nitric oxide type IIin skeletal muscle (K. D. Finley, quoted in Ref. 336); the

CAT-2/-2a isoforms are prevalent in this tissue (242). Some synthase (inducible nitric oxide synthase or NOS II), andnitric oxide production (129, 519, 612). Unpublished dataof these stress situations have a muscle proteolytic state

in common (309, 324). The possible physiological role of from MacLeod’s lab showed specific induction of mCAT-2 but not of mCAT-2a after activation of macrophagesCAT-2a upregulation induced by stress conditions such as

fasting in skeletal muscle is far from understood. MacLeod (quoted in Ref. 336). Nitric oxide synthase II uses L-argi-nine as a substrate (373), and its activity is dependent onand Kakuda (336) suggested that this is a mechanism to

prevent depletion of those amino acids from the tissue extracellular L-arginine (55, 211, 230, 611). In parallel withmacrophage activation, there is an increase in argininewith active proteolysis. In this regard, it should be men-

tioned that the rate of release of lysine or arginine in the transport rate (55, 473), due to system y/ (36). More re-cently, concomitant induction of NOS II and CAT-2 tran-rat perfused hindquarter preparation is not modified in

response to 48 h of fasting (464). Brief starvation in hu- scripts (ú20-fold) (no CAT-1 or CAT-2a transcripts) andnitric oxide production (ú30-fold) and high-affinity y/ ac-mans has been reported to enhance the release of lysine,

but not of arginine, from skeletal muscle (429). Studies tivity (3- to 4-fold) has been observed in brain astrocytestreated with LPS/IFN-g (512). Interestingly, actinomycinshould be performed to determine the role of CAT-1, CAT-

2a, and CAT-4 in the metabolism of cationic amino acids D blocks the increase in system y/ activity and nitricoxide production due to LPS/IFN-g. This indicates thatin skeletal muscle.

Ashcroft and co-workers (504) studied the transport the contribution of CAT-1 to arginine uptake for nitric


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oxide production is negligible compared with that of CAT- ing of the rest of transporters of this superfamily (forreferences for the amino acid transporters, see Table 4).2. Similar to macrophages and astrocytes, vascular

smooth muscle cells respond to cytokine (interleukin-1b The members of this superfamily have a commonmembrane topology prognosis of 12 TM domains, and asand tumor necrosis factor-a) treatment by parallel in-

crease in mCAT-2 and NOS II mRNA levels, without any a general characteristic that defines this superfamily, thetransport activity of these carriers has been found to beeffect on mCAT-1 mRNA levels (167). In contrast, angio-

tensin II increases mCAT-1 mRNA levels in vascular sodium and chloride dependent. Substitution of sodiumby lithium is not tolerated, and substitution of chloridesmooth muscle cells without induction of the nitric oxide

secretion pathway (323). No attempt was made in any of by other anions ranked the potency order as follows: chlo-ride ¢ Br0 ú NO0

3 ú gluconate ú acetate (reviewed inthese studies to corroborate changes in CAT-2 and CAT-1 expression at the protein level. All this suggests that Refs. 44, 260, 302, 465). These transporters (15 putative

cDNA have been identified without considering speciesCAT-2 transporter accounts for this increased uptake.Knockout experiments are needed to elucidate the partici- counterparts) share a high level of homology (30–65%).

Homologous transporters have been identified in insects,pation of CAT-2 and CAT-1 isoforms in the supply of argi-nine for nitric oxide synthesis by NOS II. If this hypothesis worms, and yeast (13, 318; reviewed in Ref. 390). On the

basis of substrate specificity and extensive amino acidis verified, the mechanism by which arginine supplies ni-tric oxide synthesis by NOS II will be the induction of a sequence identity, this superfamily has been divided into

two major subfamilies (reviewed in Refs. 253, 390, 565).specific y/ isoform (with transport characteristics verysimilar to the widespread y/ isoform) but through the The first major subfamily corresponds to the sodium- and

chloride-dependent neurotransmitter transporters, whichinduction of its specific gene promoter. In addition,whether differential transport properties or specific loca- comprises three minor subfamilies: 1) biogenic mono-

amine transporters, which include dopamine, norepineph-tion in plasma membrane domains of CAT-1 and CAT-2isoforms channel arginine for NOS II has not been ad- rine, and serotonin transporters that show an amino acid

sequence identity between 40 and 47% with the membersdressed. An interesting experiment would be to induceNOS II activity in the context of knockout of CAT-2 gene of the other subfamilies (53, 213, 279, 405, 494, 567, 570;

for review, see Refs. 54, 253, 390, 565) and two subfamiliesand overexpression of CAT-1 gene.for amino acid transporters; 2) GABA and taurine trans-porters (see Tables 4 and 5), also including creatine trans-

B. Superfamily of Sodium- and Chloride- porters (2 have been isolated, one of which correspondsDependent Neurotransmitter Transporters to the previously identified choline transporter as revealed

by expression studies and cellular distribution; Refs. 34,172, 186, 346, 388, 480, 509), with an amino acid sequenceThe cloning of rat and human brain GABA transport-

ers (GAT1) in 1990 (184, 391) and the human norepineph- identity that ranges between 49 and 69%; and 3) glycineand proline transporters that show a similar level of ho-rine transporter (NET) in 1991 (405) was the starting point

for the isolation of related cDNA that constitute the sodi- mology with each other and with the rest of the superfam-ily (amino acid sequence identity ranges from 43 to 48%).um- and chloride-dependent neurotransmitter transporter

superfamily (for review, see Refs. 54, 253, 390, 565). Rat The second major subfamily comprises three ‘‘orphantransporters’’ with structural characteristics that differbrain GABA transporter was purified and microsequenced

by Kanner’s group (439), and then related oligonucleotide from the previous major subfamily (large second andfourth putative extracellular domains with a canonical N-probes were used, in a collaborative effort between Nel-

son’s, Lester’s, and Kanner’s groups, to isolate rat GAT1 glycosylation site; Refs. 139, 317, 345, 566; for review, seeRef. 565).from a rat brain cDNA library (184). Amara’s group (405)

isolated the NET cDNA from the human neuroblastoma In the present review, only the amino acid transport-ers of this superfamily are considered. Excellent reviewscell line SK-N-SH by expression cloning, after accumula-

tion of the norepinephrine analog [125I]iodobenzylguanid- concerning the monoamine, creatine, and orphan trans-porters are available (11, 22, 54, 302, 565). Because of theine in COS-1 cells (405). In 1992, Burg and Handler and co-

workers (614) reported the isolation of the canine betaine high level of confusion with different names for the sametransporter cloned from different species (e.g., see differ-transporter (BGT-1) cDNA, which was obtained by ex-

pression cloning in oocytes from a size-fractionated cDNA ent names for the GABA transporters in rat and mouse inTable 4), in the present review we use the nomenclaturelibrary constructed from MDCK cells maintained in hyper-

tonic medium; mRNA from MDCK cells maintained in hy- based on the rat GABA transporter cDNA (see Table 4).In this section, attention is paid to tissue distribution,pertonic medium showed increased induction of betaine

uptake in oocytes (452). Different strategies based on ho- transport characteristics, protein structure with relationto transport function, and the physiological role of thesemology with these previous and the on-coming cDNA se-

quences from different cDNA sources resulted in the clon- transporters in both neural and peripheral tissues. The


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TABLE 4. Sodium- and chloride-dependent amino acid transporters within the superfamily

of neurotransmitter transporters

ProteinTransporters Accession Numbers Human Amino Acid(Gene Name) (Origin of Human Clones) Origin of First Clone and Other Names Chromosome Length

GAT-1 (SLC6A1) X54673 (brain) (391) Rat brain (GAT-1) (184) 3p24-p25 (220) 599Mouse GABAT (318), rat GAT-A (103)

GAT-2 (human not M95762 (rat brain) (60) Rat brain (GAT-2) (60) 602available) Mouse GAT3 (315)

GAT-3 (not shown S75989 (fetal brain) (57) Rat brain (GAT-3, GAT-B) (60, 103) (?) 632in GDB) Mouse GAT4 (315)

BGT-1 (not shown L42300 (brain) (59) MDCK cells (BGT-1) (614) 12p13 (446) 614in GDB) U27699 (kidney) (446) Mouse GAT2 (322)

TAUT (SLC6A6) Z18956 (thyroid cells) (232) MDCK cells, rat, and mouse brain (TAUT, 3p24-p26 (445) 620U16120 (placental cells) (445) Tau) (316, 503, 562)U09220 (retinal epithelium) (368)

GLYT1 (SLC6A9) P48067 (brain) (282) Rat and mouse brain (GLYT, GLYT1) (183, 1p31.3-p32 (282) 633 (1a)319, 502) 638 (1b)

Rat GLYT2 (62) 692 (1c)GLYT 1a, 1b & 1c variants (2, 62, 282, 314)

rGLYT2 (human not L21672 (rat brain) (314) Rat brain (GLYT2) (314) 799available)

PROT (SLC6A7) Not available (brain) (489) Rat brain (PROT) (155) 5q31-q32 (489) 636

Human transporters with accession numbers for their cDNA are indicated when available. Human GAT-2 and GLYT2 cDNA have not beenisolated; in their place, rat cDNA are indicated. For GLYT1, protein length is indicated for each variant (1a, 1b, and 1c). GDB, Gene Data Bank;r, rat. Reference numbers are given in parentheses.

amino acid transporters of this superfamily of neurotrans- processes of glial cells in the cerebral cortex, cerebellum,hippocampus, and retina (364, 375, 442, 450). Similarly,mitter transporters do not necessarily have a role in neu-

rotransmission, since, for example, the GABA transporter both in neuronal and type 2 astrocyte cultures, most ofGABA transport (Ç75%) shows a pharmacological profile(GAT-2), the betaine/GABA transporter (BGT-1), the tau-

rine transporter (TAUT), and the glycine transporter specific for GAT-1 (e.g., inhibition by the GAT-1-selectiveligand NNC-711) and in parallel GAT-1 transcript levelsGLYT1 are expressed in nonneural tissues (see Table 5).are abundant (61). The distribution pattern of GAT-1makes it a good candidate for the GABA transporter that1. Tissue expressionfunctions in the GABAergic synapses (see review in Ref.

The tissue distribution of GAT-1 was monitored by 240). Thus immunofluorescence, electron microscopy,immunocytochemistry before the ‘‘cloning era’’ of these and confocal immunolocalization of GAT-1 in rat hippo-transporters (442). For the rest of the amino acid trans- campus, retina, and cerebellum correlate with its involve-porters of this superfamily, the tissue distribution and the ment in the termination of the action of GABA by itstranscript size were initially studied by Northern blot (see uptake from the extracellular space into GABAergic axonTable 5) and in situ hybridization analysis. All these trans- terminals and astrocytes, where it could also play theporters are expressed in the central nervous system additional role of regulating the extracellular concentra-(CNS), and some of them are also expressed in peripheral tion of GABA (216, 375, 450). In addition, there is generallytissues. The GABA transporters GAT-1 and GAT-3, the good correlation between GAT-1 (mRNA and protein)glycine transporter GLYT2, and the proline transporter and glutamic acid decarboxylase-like immunoreactivityPROT seem to be specific to the CNS (57, 103, 155, 184, (GAD67; i.e., the enzyme responsible for GABA synthesis)240, 314, 315, 391, 489). in striatum and cerebral cortex (24, 364, 525). However,

A) GABA TRANSPORTERS. Several studies address the the expression of GAT-1 is not restricted to neurons in-distribution of the four GABA transporters (GAT-1, -2, volved in GABAergic synapses. Thus, in cerebral cortex,and -3 and BGT-1) in the CNS, including a recent review the GAT-1 uptake system (mRNA and protein) is moreby Borden (56). The three high-affinity (Km values from 1 extensive than the GABA synthesizing system (364, 525)to 20 mM) GABA transporters GAT-1, -2, and -3 are ex- in the cerebellar cortex, GAT-1 and GAD67 mRNA do notpressed in brain and retina (60, 65). Antibodies raised to correlate in Purkinje cells (447), and in the rat spinalmouse and rat GAT-1 showed a relatively even distribu- motor neurons, there is expression of GAT-1 mRNA buttion of this GABA transporter in all parts of the brain, in not GAD67 (506). All these results support the hypothesisparallel with the distribution of GABA (330, 442). This that GAT-1 plays its presynaptic role in GABAergic syn-

apses but, in addition, may have postsynaptic roles, andtransporter is present mainly in the neuropil, but also in


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TABLE 5. Tissue distribution and transport characteristics of expressed Na/- and Cl0-dependent

amino acid transporters

Tissue DistributionTransporter (Transcript Size) Expression System Substrates (Km, mM) Pharmacology (Ki, mM) Cotransported Ligands

GAT-1 CNSa (4.2–4.4 kb) Oocytesh GABA (õ10) NIP (õ10), ACHC (Ç100) GABA, 2Na/, Cl0 (265,COS-7 cells (60) L-DABA (õ30) 273)HeLa cellsi Betaine (úú500)HEK 293 cells (81, b-Ala (ú2,000)

82)GAT-2 Peripheral tissues and Oocytes (315) GABA (Ç20) NIP (ú500), ACHC (ú500) Na/, Cl0 (60)

CNSb (2.2–2.4 kb) COS-7 cells (60) b-Ala (Ç30) L-DABA (ú100), betaineTaurine (Ç500) (úú500), b-Ala (Ç100)

GAT-3 CNSc (4.7–5.0 kb) Oocytes (315) GABA (Ç1) NIP (50), ACHC (Ç800) GABA, ú1Na/, Cl0

COS-7 cells (60) b-Ala* (Ç100) L-DABA (Ç100) (102)LLC-PK1 cells (102) Taurine (Ç1,500) Betaine (úú500)Intestine cells (103) b-Ala* (õ10)

BGT-1 Peripheral tissues and Oocytes (614) GABA (õ100) NIP (ú2,000), ACHC GABA, 2Na/, Cl0 (194)CNSd (3.0–3.4 kb COS-7 cells (59) Betaine (Ç400) (Ç2,000)and others) 9HTEo cells (446) L-Proline (Ç900) L-DABA* (¢2,000)

LM(tk-) cells (59) Betaine* (Ç200)MDCK cells (423) b-Ala (Ç2,000)

TAUT Peripheral tissues and Oocytesj Taurine (õ10) Hypotaurine (õ10) Tau, 2Na/, Cl0 (445)CNSe (6.2–7.5 kb COS-7 cellsk b-Ala (Ç60) L-Ala (ú250), L-Pro (úú250)and others) HeLa cells (445) GABA* (ú2,000)

MeAIB (úú2,000)GLYT1 CNS and peripheral Oocytesl Glycine (õ50) Sarcosine (õ100) Na/, Cl0 (183, 282,

tissuesf (3.2–3.8 kb) COS-7 cellsm L-Ala, GABA (ú1,000) 319)L-Pro (¢1,000)

GLYT2 CNS (314) (8 kb) Oocytes (22) Glycine (Ç20) Sarcosine (ú1,000) Nap, Cl0 (314)L-Ala, MeAIB (ú1,000)b-Ala (ú2,000)

PROT CNSg (4 kb) HeLa cellsn L-Pro (õ10) Sarcosine (30), NIP (úú100) Na/, Cl0 (489)L-His, L-Cys (Ç80)

Only range of common transcript size in different species is shown. Km values are from different labs and obtained by expression in Xenopus

oocytes. * Substrates or inhibitors with contradictory reports for interaction with corresponding transporter from different labs or species (seetext for details). All transporters have been shown to cotransport corresponding substrates plus co-ions, Na/ and Cl0. In addition to biogenicamine transporters of this superfamily, GAT-1 transporter has been shown to have ion leak and ligand-gated channel activity (see text fordetails); there are no reports on this issue for the rest of the amino acid transporters of this superfamily. NIP, nipecotic acid; ACHC, cis-3-aminocyclohexanecarboxylic acid; L-DABA, 2,4-diaminobutyric acid; b-Ala, b-alanine; L-Ala, L-alanine; MeAIB, N-methylaminoisobutyric acid; L-pro, L-proline; sarcosine, N-methylglycine; L-His, L-histidine; L-Cys, L-cysteine. References are as follows: a) 184, 315, 391; b) 60, 315; c) 60, 103,315; d) 59, 322, 446, 614; e) 232, 316, 368, 445, 503, 562; f) 2, 62, 183, 282, 319, 502; g) 155, 489; h) 184, 265, 318, 325, 339, 341, 520; i) 37, 261, 273,288, 410, 451; j) 316, 368, 562; k) 232, 503; l) 183, 319; m) 62, 282, 502; n) 155, 489. Other references are as given in parentheses.

it may regulate the extracellular concentration of GABA sively to astrocytic processes (363). Similarly, GAT-3mRNA is present in neuronal and in type 2 astrocyte cellin glial cells.

Northern blot analysis and in situ hybridization histo- cultures (61). Recently, in electron microscopic immuno-localization studies in the developing rat brain (i.e., em-chemistry showed a complementary distribution of GAT-

3 and GAT-1 (103, 315); GAT-3 has a strong expression in bryonic and early postnatal stages), GAT-1 and GAT-3protein showed coordinate expression (239): GAT-1 wasspinal cord, brain stem, thalamus, and hypothalamus and

is weakly expressed in cerebellum, hippocampus, cere- detected in gray matter and growing axons, and GAT-3 inradial glial cell fascicles oriented perpendicularly to thebral cortex, and striatum, where GAT-1 transcripts are

abundant. In many instances, the localization of GAT-3 axons expressing GAT-1. Therefore, at present, it seemsthat GAT-3 is mainly a glial transporter.(mRNA and protein) correlates well with characterized

populations of GABAergic neurons and glutamic acid de- Among the high-affinity GABA transporters, GAT-2 isthe only one expressed in peripheral tissues in additioncarboxylase immunoreactivity, as in the medial septum-

diagonal band complex, but in others, the distribution is to brain and retina (60, 223, 315). Initial Northern blotanalysis revealed that GAT-2 mRNA levels in brain aredissimilar, such as in the cerebellum (103) and cerebral

cortex (363). The distribution of GAT-3 in brain cells is developmentally regulated, being more abundant in thebrains of newborn mice than in the adult or fetal braincontroversial. Initially, GAT-3 was described predomi-

nantly as a neuron transporter (103), but later its presence (315). Two studies compared the distibution of GAT-2with the other two high-affinity GABA transporters in ret-in astroglial processes was demonstrated by immunocyto-

chemistry (450), and in cerebral cortex it localized exclu- ina and brain (216, 223). Immunoreactivity of GAT-2 was


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faint throughout the brain but was concentrated in the MDCK cells; expression of BGT-1 in hippocampal neu-rons in culture localized to somatodendritic membranes.arachnoid and ependymal cells, a completely different dis-

tribution from that of GAT-1 and GAT-3 proteins (see These studies are subsidiary to those of Simons and co-workers (130, 131, 221) showing a polarized correlationabove). Similarly, in the retina, GAT-2 protein localizes to

the retinal pigment epithelium layer, nerve fiber layer, between axons and the epithelial apical pole, and so-matodendritic membranes and epithelial basolateraland cilliary body epithelium, whereas GAT-1 and GAT-3

localize to amacrine neurons and Muller glial cells, respec- membranes. Following this line, Caplan and co-workers(5) suggested that GAT-2 may have a basolateral loca-tively. In rat brain-derived cultures containing O-2A pro-

genitor cells and type 2 astrocytes, GAT-2 mRNA is the tion in kidney and liver and a dendritic location in neu-rons, but to our knowledge, the subcellular distributionsecond most abundant after GAT-1 transcripts; in con-

trast, GAT-2 transcripts are not detected in neuronal cul- of GAT-2 in epithelia and brain has not been determined.Very recently, Pietrini, Caplan, and co-workers (417),tures or type 1 astrocyte cultures (61). These results sug-

gest that GAT-2 may be related to nonneuronal function working with MDCK cells BGT-1, rat GAT-1 and humannerve growth factor receptor chimeras, suggested thein brain and retina.

Northern blot analysis showed an even distribution presence of basolateral sorting information in the cyto-solic COOH-terminal domain of MDCK cells BGT-1; aof BGT-1 transcripts in mouse (Ç5 kb in length) and hu-

man brain (¢3 kb and a less conspicuous band of 4.1 kb) short segment within this domain (residues 565–572),rich in basic residues well conserved in human BGT-1(59, 322, 446). BGT-1 is most probably a glial transporter;

its transcripts were observed in type 1 and type 2 but not in rat GAT-1, contains information necessary forexit from the endoplasmic reticulum and for the basolat-astrocyte cultures, but not in neuronal cell cultures (61).

The presence of BGT-1 mRNA in human and mouse brain eral localization of MDCK cells BGT-1 in these cells.B) TAURINE, GLYCINE, AND PROLINE TRANSPORTERS. To(59, 322, 446) suggests that, at least in these species (BGT-

1 was not shown in canine brain; Ref. 614), this trans- our knowledge, the tissue distribution of the taurinetransporter TAUT has been studied only by Northernporter could participate in brain osmoregulation (59) (see

sect. IIB6). Betaine is present in brain at low concentra- blot analysis or RT-PCR. The TAUT transcripts arewidespread, and its abundance varies between differenttions, but levels increased after salt loading (207). Han-

dler’s group (537) showed the presence of different 5*- studies and species. In general, however, it has beenshown to be present in kidney cortex and medulla, smallUTR in canine BGT-1 transcripts due to splice variants

and the use of three tissue-specific promoters. intestinal mucosa, brain, lung, retina, liver, skeletalmuscle, heart, placenta, spleen, and pancreas (232, 316,The GABA transporters GAT-2 and BGT-1 are ex-

pressed in peripheral tissues (59, 60, 315, 322, 446, 614): 445, 503, 562). Reverse transcriptase-polymerase chainreaction also showed TAUT mRNA in human ovary, co-mouse and rat GAT-2 are expressed in liver and kidney

and human and mouse BGT-1 are expressed in kidney lon, and thyroid (232).The tissue distribution of the glycine transporters, asmedulla and liver. In addition, one report also showed

expression of human BGT-1 in placenta, heart, and skel- revealed by Northern blot analysis, indicates that GLYT1is expressed in the CNS and peripheral tissues, whereasetal muscle (446). In situ hybridization and RT-PCR of

microdissected nephron segments revealed that BGT-1 GLYT2 is specific to the CNS (see Table 5). A recent re-view by Zafra et al. (622) summarizes the cellular localiza-is predominantly expressed in the medullary thick as-

cending limbs of Henle’s loop and the inner medullary tion studies of glycine transporters at the protein andmRNA levels in brain (2, 62, 183, 237, 314, 328, 502, 623,collecting ducts (365). In the polarized epithelial cell

model MDCK, Handler’s group (613) showed that BGT- 625). Both GLYT1 and GLYT2 are mainly expressed incaudal areas, and in addition, GLYT1 has a moderate ex-1 localizes to the basolateral membrane, consistent with

its role in protecting cells in the renal medulla from pression in forebrain areas. The distribution of GLYT2 isconsistent with the distribution of the inhibitory glycinehypertonicity (385, 387). Caplan and co-workers (5, 423)

addressed the polarized expression of the four GABA receptor (immunocytochemistry and strychnine bindingstudies) and with neurons with high glycine content (17,transporters (GAT-1, -2, and -3 and BGT-1) expressed in

MDCK and in freshly isolated hippocampal neurons: 1) 237, 623): high expression in the dorsal and ventral horn ofthe spinal cord, auditory system, and nuclei of the cranialGAT-1 localized exclusively to the axons of cultured

neurons and to the apical pole of transfected MDCK nerves, and low expression in cerebral hemispheres. Thedistribution of GLYT1 in caudal areas is more widespreadcells, consistent with its axonal localization in vivo (364,

375, 442, 450). 2) GAT-3 was expressed in the apical than that of the glycine receptors detected by strychninebinding (623). This indicates first, association of bothmembrane of transfected MDCK cells and in both axons

and somatodendritic membranes of hippocampal neu- transporters with the inhibitory glycinergic neurotrans-mission and a role in the termination of the glycinergicron cultures. 3) In contrast, expressed GAT-2 and BGT-

1 localized to the basolateral membranes of transfected action, and second, additional roles for GLYT1 in nongly-


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cinergic regions. Electron microscopic immunolocaliza- the amino acid transporters of this superfamily are sum-marized in Table 5. Sodium and chloride dependence hastion showed a glial (perikarya and processes) location for

GLYT1 and an axonal (presynaptic terminals) location for been reported for all these transporters (see Table 5).B) GABA TRANSPORTERS. Of the eight amino acid trans-GLYT2 (623). The location of GLYT1 in neurons is at pres-

ent controversial: some authors did not observe neuronal porters in this superfamily, only four transporters, GAT-1, -2, and -3 and BGT-1, induce uptake of GABA whenexpression of GLYT1 mRNA (2, 183), whereas others (62,

502, 625) reported in situ hybridization signal in neurons expressed in cultured cells or in oocytes; GAT-1 to -3 arehigh-affinity transporters (Km values from 1 to 20 mM),of the spinal cord, brain stem, and cerebellum and also

in forebrain regions (cortex, hippocampus, thalamus, hy- and BGT-1 is a low-affinity GABA transporter (when ex-pressed in oocytes it showed an apparent Km ofõ100 mMpothalamus, and olfactory bulb). Immunocytochemistry

studies failed to detect the neuronal form of the protein for GABA, even lower than that for betaine) (see Table5). Pharmacological studies helped to distinguish theclearly (except in the retina; GLYT1 protein was localized

in the amacrine neurons, Ref. 623), but they confirmed transport activity of the four GABA transporters (see Ta-ble 5). GAT-1 shows the highest sensitivity to cis-3-amino-the glial expression of GLYT1 (238, 623). This controversy

might be because of the use of probes with different iso- cyclohexanecarboxylic acid (ACHC), 2,4-diaminobutyricacid (L-DABA), nipecotic acid (NIP) (also OH-NIP), andform specificity and antibodies that do not react with the

neuronal form of the protein (622). The specific mRNA guvacine, and it is not inhibited by b-alanine (60, 103, 117,273, 315). These are the pharmacological characteristicsdistribution of GLYT1–1a and 1b/1c (GLYT1–1b probes

used in this study do not distinguish between 1b and 1c of the neuronal GABA transport (see references in Refs.103, 184). In addition, biochemical evidence demonstratedvariants) isoforms has been studied by Borowsky et al.

(62): GLYT1–1a is found only in gray matter, whereas that GAT-1 corresponds to the neuronal high-affinity sub-type GABAA transporter, sensitive to ACHC (for review,GLYT1–1b/1c is found exclusively in fiber tracts. Further-

more, GLYT1–1b/1c is found in all white matter, whereas see Ref. 260). Lipophilic derivatives of piperidencarboxy-lic acid [tiagabine, SKF-89976A, CI-966, and NNC-711; theGLYT1–1a distribution parallels the distribution of mRNA

for the strychnine-sensitive and strychnine-insensitive gly- latter with an inhibition constant (Ki) of 6 nM] are highlyselective for GAT-1 transport activity (58, 102, 103). Inter-cine receptor in olfactory bulb, hippocampus, cerebellum,

and spinal cord. For the presence of GLYT1 in areas with- estingly, these inhibitors have anticonvulsant properties(103, 524).out glycine receptor expression, two explanations have

been offered (622): 1) Smith et al. (502) suggested a role of The pharmacologies of GAT-2 and GAT-3 are similarto each other; GAT-3 shows higher sensitivity to NIP (seeGLYT1 in modulating glutamatergic transmission through

the activity of some N-methyl-D-aspartate (NMDA) recep- Table 5) and to a new triarylnipecotic acid derivative, 4(S)(128). The most characteristic BGT-1 inhibitor is betainetors (see sect. IIB6), and 2) association of GLYT1 with

non-strychnine-sensitive glycine receptors; in this sense, (see Table 5). Kilimann and co-workers (187) listed thepharmacological characteristics of the GABA transportersthe mRNA of the b-subunit of the glycine receptor and

GLYT1 show similar distribution (62, 625). isolated from different species and expressed in differentsystems and pointed out the dissimilarities in the dataGLYT1 is expressed in peripheral tissues (see Table

5). The GLYT1–1a variant, but not 1b/1c variants, is reported (e.g., BGT-1 inhibition by L-DABA and betaine,TAUT inhibition by GABA, and interaction of b-alanineexpressed in rat peripheral tissues: liver, spleen ú lung

ú stomach, uterus ú pancreas, kidney (i.e., relative with GAT-3; see Table 5). One of the most relevant dissimi-larities is the role of b-alanine in GAT-3 transport activity:abundance of transcripts) (62). Expression of GLYT1–

1a in lung, spleen, liver, and thymus occurs in macro- an apparent Km of Ç100 mM for mouse GAT-4 (i.e., GAT-3) (315), and no transport of b-alanine up to 500 mM viaphages (62).

The mRNA coding for the brain-specific high-affinity L- rat GAT-B (i.e., GAT-3), but an apparent Ki of Ç7 mM forb-alanine inhibiting GABA transport (103). More recentproline transporter PROT was shown to be expressed in

subpopulations of putative glutamatergic neurons in the ol- studies (102) showed consistent interaction of GAT-3 per-manently expressed in LLC-PK1 cells with b-alanine (i.e.,factory bulb, cerebral cortex, and hippocampus (155). West-

ern blot studies revealed the presence of PROT in enriched similar apparent Km and Ki values of Ç30 mM). The rea-sons for these discrepancies are unknown. Interaction ofsynaptic plasma membrane preparations and its absence

from postsynaptic membranes (489, 580). This suggests a b-alanine with GAT-3 is consistent with its presence inglial cells, but not with its expression in neurons (61, 103,presynaptic regulatory role of L-proline or the PROT trans-

porter in specific excitatory pathways in the CNS. 216, 363, 450).In summary, the pattern of expression in brain and

2. Transport characteristics the pharmacological characteristics of the four GABAtransporters strongly indicate that GAT-1 corresponds toA) GENERAL CHARACTERISTICS. The characteristics of

the transport activity associated with the expression of the most typical neuronal presynaptic GABA transporter,


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whereas GAT-2 and GAT-3 characteristics fit the glial amino acid sequence identity ranges from 62 to 66%;amino acid residues present in b-amino acid interactingtransporter activity. In addition, the neuronal expression

of GAT-3 and its brain distribution implies a complemen- transporters: GAT-2, GAT-3, TAUT, and BGT-1 are idi-cated in Fig. 3). As for the taurine transporter, GAT-2tary role of GAT-3 and GAT-1 in GABAergic synapses. The

b-alanine sensitivity of neuronal GAT-3, a glial character- and GAT-3 transport activity is inhibited by micromolarconcentrations of hypotaurine (562). Therefore, the sub-istic (103), might be explained by this complementary

expression of GAT-1 and GAT-3, and the higher expres- strate specificity of GAT-2 and GAT-3 transporters coversGABA and the b-amino acids. As indicated above, GAT-2sion of GAT-1 in neurons, as described in neuronal cell

cultures (61). and GAT-3 represent tissue/cell-specific isoforms withvery similar transport activity: GAT-3 is neural tissue spe-C) b-AMINO ACID TRANSPORTERS. Four transporters of

this superfamily mediate b-amino acid transport (GAT-2, cific, whereas GAT-2 is highly expressed in kidney andliver, and in lower amounts in adult brain.GAT-3, and TAUT) or are inhibited by b-alanine (BGT-1)

(see Table 5). Murine, canine, rat, and human TAUT are BGT-1, with high homology (amino acid sequenceidentity ranges from Ç60 to 70%) to the b-amino acidhighly homologous (Ç90% identity), have a wide tran-

script distribution, including kidney and small intestine, transporters (i.e., TAUT, GAT-2, and GAT-3), encodes fora sodium- and chloride-dependent betaine (fully methyl-and express a very similar transport activity for the sulfur-

containing b-amino acids taurine and b-alanine in oocytes ated glycine) and GABA transporter (see sect. IIB2B). In-terestingly, b-alanine interacts weakly with BGT-1 (i.e., 2(232, 316, 368, 445, 503, 562). Apart from the difference

in the length between the mouse (590 amino acid residues; mM b-alanine inhibits Ç50% of transport). BGT-1 cDNAfrom mouse, dog, and human most probably representsRef. 316) and the human, rat, and canine proteins (620,

621, or 655 amino acid residues, respectively; Refs. 232, species counterparts of the same transporter because oftheir homology (Ç90% amino acid sequence identity) and368, 445, 562), because of different COOH termini, all

these transporters could be considered as the counter- similar transport characteristics (see Table 5), in additionto their reported different tissue distribution in brain andparts of the same gene for these species. Expression of

these transporters in oocytes results in sodium- and chlo- peripheral tissues (see above). The murine and humanBGT-1 are expressed in kidney, liver, and brain, but theride-dependent high-affinity transport of taurine (Km, 5–

12 mM) and b-alanine (Km, Ç50 mM); hypotaurine inhibits canine transporter is mainly expressed in kidney medullabut not in liver or brain (59, 322, 446, 614). Most probably,transport with a Ki that is probably in the micromolar

range, whereas GABA, L-alanine, and MeAIB have no ef- the canine BGT-1 transporter represents the major hyper-tonicity-modulated transporter of the nonperturbing os-fect at 100 mM (see Table 5). Thus it seems that the tau-

rine/b-alanine transporter is a b-amino acid-specific car- molyte betaine that operates in kidney medulla, normallythe only hypertonic tissue in mammals (292). As discussedrier. A specific system for b-amino acids with high affinity

for taurine (Km, 10–14 mM) and similar transport charac- by Yamauchi et al. (614), luminal membranes from smallintestine and renal proximal tubules transport betaine interistics has been described in luminal plasma membranes

from small intestine and renal proximal straight tubules; a sodium-dependent manner, which is shared with high-affinity transport of proline. Interestingly, canine BGT-1furthermore, in renal luminal membranes, other transport

systems with lower affinity (Km values in micromolar and transporter expressed in oocytes has a low affinity forproline (Km in the millimolar range) (614).millimolar ranges) for taurine have been reported (231,

370, 496). A similar transport activity has been reconstitu- In summary, transporters of this superfamily can begrouped according to substrate specificity. From strictted from brush-border membranes of human placenta

(444). Handler and co-workers (562) proposed that the GABA transporters to strict b-amino acids (i.e., b-alanine)transporters, the proteins could be ranked as follows (seecloned taurine carrier may correspond to the basolateral

transporter described in MDCK cells, since the levels of Table 5): 1) GAT-1, high affinity and GABA specific; nointeraction with b-alanine; neural specific; 2) BGT-1, inhi-the carrier mRNA increase after incubation in hypertonic

medium. However, this issue is not clear, since the Km bition by b-alanine at millimolar concentration; 3) GAT-2 and the neural-specific GAT-3, substrate affinity in the(Ç50 mM) reported for the basolateral carrier in MDCK

cells is higher than that reported for the cloned carrier micromolar range for GABA and b-alanine; and 4) TAUTtransporter, substrate affinity in the micromolar range forand for the apical membranes of MDCK cells (Ç10 mM)

(563). Both GAT-2 and GAT-3 encode for high-affinity b-alanine and taurine and weak, if any, interaction withGABA. The concept of classical system BETA, defined inGABA transporters (see above). These two transporters

also carry b-alanine with high affinity (Km values are Ç30 Ehrlich ascites cells with a low affinity for b-alanine (Km

in millimolar range) (96, 495), might correspond to theand Ç100 mM, respectively) and taurine with a lower af-finity (Km values are Ç500 and Ç1,500 mM, respectively) variety of different transporter isoforms described here

and to carrier isoforms not yet identified. For example,when expressed in oocytes. Homology between these twotransporters and the taurine transporter is very high (i.e., in mammalian kidney, a complex interaction between re-


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absorption systems for b-amino acids and GABA has been and is mainly found in neural tissues with very low levelsof mRNA in liver or kidney (2, 62, 183, 282, 319, 502). Thusshown, including shared transport systems for amino

acids and GABA as well as more GABA-specific carriers the encoded protein might correspond to a transporterisoform of the widespread system Gly. GLYT1 gene ex-(496). TAUT, BGT-1, GAT-2, and GAT-3 transporters,

which show overlapping specificities for b-amino acids pression (1a variant; see sect. IIB1) in kidney, althoughlow, might be responsible for the high-affinity (Km in theand GABA, are expressed in kidney. The challenge is now

to relate the function of these transporters with the physi- micromolar range) sodium-dependent glycine uptakecomponent in luminal membranes from proximal straightological fluxes of these amino acids in vivo, through the

regional and cellular localization of these transporters and tubules (reviewed in Ref. 483). This can now be tested byimmunolocalization of the GLYT1 in kidney. To this end,the ‘‘knockout’’ of the respective genes.

D) GLYCINE TRANSPORTERS. Two glycine transporter it is important to assess whether GLYT1 in kidney is ex-pressed in the epithelial cells or in macrophages, as hasgenes, GLYT1 and GLYT2, belong to this superfamily (see

Table 5). GLYT1 presents three variants (1a, 1b, 1c) that been reported for lung, spleen, and liver (62).E) PROLINE TRANSPORTER. The proline transportersare transcribed from a single gene (2, 62, 282). The three

protein variants differ in their NH2-terminal sequences. (PROT) isolated from a rat and human brain librariesshow Ç45% identity to GLYT transporters at the aminoTwo promoters are responsible for 1a and 1b variants,

and 1c isoform is an alternative splicing variant of 1b acid sequence level (155, 489). The protein is encoded bya 4-kb mRNA that is present in the excitatory pathwaystranscript with a 54-amino acid-long exon toward the NH2

terminus (2) (see Fig. 4B). The three variants show no of rat brain. This pattern of expression suggests that theencoded protein does not represent an ubiquitous trans-differences in their transport characteristics; in fact, trun-

cated proteins, constructed by elimination of the differen- porter that might have a general metabolic role but rathersupports a specific role for L-proline or this transportertial amino acid residues, retain the transport characteris-

tics of the intact proteins, and only their cellular pro- in excitatory amino acid neurotransmission (see sect.IIB6). The transporter expresses sodium- and chloride-cessing is affected (282). GLYT2 (Ç50% amino acid

sequence identity with GLYT1 variants) most probably dependent uptake of L-proline with very high affinity (Km

is Ç10 mM) in HeLa cells (155, 489). The expressed so-corresponds to the 100-kDa reconstituted and purified gly-cine transporter from pig brain (314, 320). GLYT2 is a dium-dependent uptake of proline is sensitive to sarcos-

ine, norleucine, phenylalanine, histidine, and cysteine,larger protein than GLYT1 variants, due to a longer NH2

terminus (see Fig. 3). GLYT1 and GLYT2 transport activi- with Ki ranging from 30 to 90 mM. Therefore, in commonwith GLYT transporters, PROT transporters tolerate N-ties can be distinguished by the higher sensitivity of

GLYT1 variants to sarcosine (N-methylglycine) (see Table methyl derivatives, since both show high-affinity interac-tion with sarcosine (N-methylglycine). This high-affinity5). Specific high-affinity (apparent Km values from 20 to

100 mM) transport systems for glycine have been identified L-proline transport is unique to nervous tissue; in contrast,a lower affinity, sodium-dependent L-proline transport hasin synaptosomes and glial cells (for review, see Ref. 622).

The uptake process is electrogenic (inward positive also been described in neural tissues and renal, intestinal,and choroid plexus brush-border membrane vesiclescharge flux) with a stoichiometry of 1 glycine, 2 sodium,

and 1 chloride (16, 624); these coupling coefficients permit through various systems (e.g., IMINO, Bo,A) (reviewed inRefs. 483, 513, 505).a glycine gradient through the plasma membrane that is

strong enough to maintain an extracellular glycine con-centration of 0.2 mM (23). The characteristics (substrate 3. Mechanisms of transport and uncoupled ion fluxesaffinity, sodium and chloride dependence, and pharmacol-ogy) of the glycine transport via GLYT1 and GLYT2 in The transport characteristics of the first member of

this superfamily of transporters to be discovered, GAT-oocytes and mammalian cells are consistent with the neu-ronal and glial transport activity (622). Because of their 1, have been studied more extensively than the other

members (see Table 5). As discussed above, GAT-1 cor-brain distribution, GLYT2 most probably represents theneuronal glycine transporter, and GLYT1 the glial trans- responds to the neuronal high-affinity ACHC-sensitive

GABA transporter present in synaptosomes and synapticporter, but the presence of GLYT1 transcripts in neurons(see sect. VB1) suggests its contribution to neuronal gly- plasma membranes (260, 273). The transport of GABA

by GAT-1 in different expression systems, including per-cine uptake.The transport characteristics of GLYT1 (sodium-de- manent expression in mammalian cells, showed the fol-

lowing characteristics (for references, see Table 4): 1)pendent glycine transport inhibited by sarcosine, but notby MeAIB or b-alanine) resemble that of system Gly (274, a single high-affinity (Km ofÇ4 mM) component of trans-

port, in full agreement with values obtained in synaptic572). Notwithstanding, the expressed transport activity ofGLYT1 presents a much higher affinity for glycine than plasma membranes (250) and reconstituted systems

(439); 2) absolute requirement of sodium, but not ofthe classical transport system (62, 183, 282, 314, 319, 502)


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FIG. 3. Amino acid sequence comparisons of sodium- and chloride-dependent amino acid transporters. Sequencesmultialigned correspond to human transporters but for rat GAT-2 and GLYT2. Horizontal lines over sequences indicateroughly position of theoretical (i.e., hydrophobicity plots) TM domains I-XII. Potential N-glycosylation sites are indicatedby open boxes between TM domains III and IV. Gray boxes indicate amino acid residues present in all mammalianamino acid transporters (negative or positive charged residues are considered together) of this superfamily, in 4 GABAtransporters (GAT-1, GAT-2, GAT-3, and BGT-1), in b-alanine-interacting transporters (GAT-2, GAT-3, BGT-1, and TAUT),or in GABA, betaine, and taurine transporter subfamily. Finally specific amino acid residues for glycine and prolinesubfamily of transporters are shown in white inside black boxes. For all amino acid sequence identity shown here, allmammalian cDNA clones indicated in Table 4 are considered. Dash lines indicate gaps for sequence multialignmentobtained with Clustal Multiple Sequence Alignment from Baylor College of Medicine.

chloride; capacitive charge movement and transport-as- the chloride concentration (i.e., a decrease in chlorideconcentration results in an increase in the apparent Kmsociated current studies indicate that chloride facilitates

the binding of sodium and that this limits the overall for GABA).Stoichiometry of the coupled ions and GABA viatransport rate at saturating GABA concentrations (339,

325); and 3) exchange mechanism of transport (i.e., ef- cloned GAT-1 was first deduced from the correspondingHill equations. GAT-1 permanently expressed in mouseflux of GABA is trans-stimulated by external GABA).

This represents a part of the translocation cycle of the LtK0 cells (273) shows a sodium concentration depen-dence with sigmoidal behavior, whereas the dependencetransporter (257, 393). Stable expression in mammalian

cells revealed unique properties for GAT-3 (102), a Km of GABA and chloride concentrations was hyperbolic.This allowed a proposed stoichiometry of cotransport ofvalues for chloride of 78 mM, which is severalfold higher

than that for GAT-1, and the Km for GABA depends on 1 GABA, ú 1 (probably 2) sodium, 1 chloride, in accor-


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FIG. 3—Continued

dance with data obtained previously with synaptic plasma transporters of this superfamily; a stoichiometry of 1 sero-tonin, 1 sodium, and 1 chloride for the rat serotonin trans-membrane vesicles and the purified transporter (257, 272,

440). Thus direct measurements of 22Na/, 36Cl0, and porter SERT (180) and 1 norepinephrine, 1 sodium, and1 chloride for the human transporter NET (161, 181).[3H]GABA fluxes using proteoliposomes into which a par-

tial purified preparation of the neuronal high-affinity Therefore, as discussed below for the sodium- and potas-sium-dependent transporters of glutamate and zwitter-ACHC-sensitive GABA transporter (most probably GAT-

1; see above) was reconstituted yielded the following stoi- ionic amino acids, the stoichiometry of the coupled ionsdoes not appear to be conserved between the memberschiometry (272): 1 GABA ¢ 2 sodium, 1 chloride. This

stoichiometry suggests that GABA transport through of the superfamily of sodium- and chloride-dependentneurotransmitter transporters.GAT-1 is electrogenic (i.e., external GABA induces posi-

tive inward current). Expression of rat GAT-1 in oocytes The transport stoichiometry of the neurotransmit-ters and co-ions just described leads to electrogenicresulted in electrogenic uptake of GABA, with an apparent

affinity constant of Ç5 mM for GABA and Hill coefficients transport. However, the conducting properties of thesetransporters go well beyond this (301, 507). The expres-of 0.7 for chloride and 1.7 for sodium (265). Concentration

dependence studies of capacitive charge movements sup- sion of these transporters displays several conductingstates that resemble single-channel openings and thatport the interaction of two sodium with GAT-1 (339). Sur-

prisingly, correlation of induced currents and radiolabeled occur both in the presence of substrate (substrate-gatedchannel activity) and in its absence (leakage) (301, 507).GABA uptake gave a ratio of one positive charge flux per

molecule of GABA transported; this indicates a stoichiom- Uncoupling between transport flux and substrate-evoked current in steady state has been observed foretry of 1 GABA, 2 sodium, and 1 chloride (265, 341). Simi-

larly, kinetics of GABA uptake induced by rat GAT-1 trans- rat SERT, human DAT, human NET, and rat GAT-1 (160,161, 340, 451). Several recent reviews address this issuefected in HeLa cells gave Hill coefficients compatible with

this stoichiometry (451). This fits with the proposed stoi- (301, 507, 609). This is similar to the uncoupled chloridechannel mode of action of several transporters of thechiometry for the rat GAT-3, canine BGT-1, and human

dopamine transporter DAT (507, 102), but not with other superfamily of sodium- and chloride-dependent gluta-


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FIG. 4A. new 12-TM domain membrane topology model for rat GAT-1 transporter. This model has been recentlyproposed by Kanners and co-workers (38), and it is in full agreement with that proposed by Aragon, Gimenez, and co-workers (401) for GLYT1 transporter (see Fig. 4B). Notice that hydrophobic domain I corresponds to a ‘‘reentrant’’membrane loop. Hydrophobic external (EL) and internal (IL) loops are numbered. A 44-amino acid hydrophobic stretch,from Leu-118 to Trp-161, is proposed to contain TM domain III, IL2 loop, and new TM domain III*; sequence withinTM domain III* is only tentative. Amino acid residues conserved in all mammalian amino acid transporters of thissuperfamily are indicated in gray circles, whereas only 6 specific amino acid residues for GABA transporters (GAT-1,-2, -3, and BGT-1) are indicated in white over a black circle. Amino acid residues within squares are those subjectedto site-mutagenesis studies (see text for details). Amino acid sequences indicated by arrows connected with a line inEL4 and EL5 are corresponding ones cross-mutated between mouse GABA transporters and proposed to be involvedin substrate specificity and affinity in Reference 542 (see text for details). Y, three putative N-glycosylation sites in EL2.Exon-intron boundaries of mouse GAT-1 transporter gene (318; corrected in Ref. 390) within putative rat proteinsequence are indicated, and exons are numbered. Exon 1 is an untranslated sequence (318). In general, position ofexon-intron boundaries is conserved between transporters of this superfamily (390) (see Fig. 4B for murine GLYT1transporter gene).

mate and zwitterionic amino acid transporters (see sect. 507). Thus, for rat SERT expressed in oocytes, a ratioof charge flux to serotonin molecule of 5–12 positiveIIC), but the charge-carrying ions responsible for these

uncoupled currents have not been identified (507). Com- charge flux was found, whereas the proposed transportstoichiometry (1 serotonin, 1 sodium, 1 chloride) pre-parisons of the transport flux and the currents associ-

ated with the expression of the biogenic amine trans- dicted a ratio of 1 positive charge flux/serotonin mole-cule (340). Electrophysiological studies with rat GAT-1porters (SERT, NET, and DAT) and the GAT-1 GABA

transporter revealed ratios greater than the proposed permanently transfected in HEK 293 cells showed thatthe stoichiometry between GABA and co-ions flux wasstoichiometries (discrepancies ranged from a factor of

3 to ú100), but the apparent Hill coefficients for sodium not fixed (e.g., outward and inward currents require dif-ferent ions on each side of the membrane) (81). DeFeliceand chloride for the activation of the substrate-induced

currents is consistent with the uptake studies and with and co-workers (451) reported that the sodium-depen-dent GABA-induced currents due to GAT-1 expressionthe proposed stoichiometry (for review, see Refs. 301,


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FIG. 4B. new 12 TM domain membrane topology for mouse GLYT1a transporter. This model has been proposedby Aragon, Gimenez, and co-workers (401) for rat GLYT1 transporter and is in full agreement with model proposed byKanner and co-workers (38) for rat GAT-1. See Fig. 4A for description of model. Amino acid residues conserved in allmammalian amino acid transporters of this superfamily are indicated by gray circles, whereas those specific for glycinetransporters are indicated in white over a black circle. N-glycosylation sites (Y) used for rat GLYT1 are indicated by asquare (see text for details). Murine exon-intron boundaries within putative protein sequence are indicated, and exonsare numbered. Inset: alignment of NH2 terminals of 3 mouse GLYT1 variants (1a, 1b, and 1c). At present, full-lengthmurine exon 1c is unknown (2), but human one has been reported (282).

in HeLa cells do not show saturation of the current- conduction, which is not thermodynamically coupled totransport. It is at present unknown, as for the membersvoltage curve, as fixed stoichiometry would predict, but

it shows saturation of the current-external GABA con- of the superfamily of sodium- and potassium-dependentglutamate and zwitterionic amino acids (see sect. IIC),centration curve at a fixed voltage; these data suggest

that saturable binding of external GABA gates a channel whether the substrate-gated channel occurs through thetransporter itself or via interaction with an extrinsicthrough the GAT-1 transporter or an associated channel.

Mager et al. (341) estimated that GAT-1 is expressed in channel protein. Fluctuation analysis indicates that thesubstrate-gated channel current events (00.2 to 00.5 pAoocytes at a plasma membrane density of 104 transport-

ers/mm2, and with a transport turnover rate of Ç10 s01 at 080 to 0100 mV), associated with the expression ofhuman NET and rat serotonin transporter (SERT) in(at 300 mM GABA, 96 mM NaCl, 080 mV and 227C). To

explain the maximal currents due to the expression of HEK 293 cells and oocytes, respectively, are brief (Ç1ms) and have an extremely low open probability (1003 toGAT-1 in HeLa cells on the basis of the 1 GABA, 2 so-

dium, 1 chloride-coupled model, either the density of 1006); interestingly, the probability of opening increaseswith substrate concentration (160, 507). This is interpre-transporters at the cell surface or the turnover rate is

ú10-fold higher than the values estimated in oocytes ted, at least for several members of the superfamily ofneurotransmitter transporters (SERT, NET, DAT, and(451). In summary, all this suggests substrate-activated


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GAT-1), as indicating that these transporters show brief of rat GAT-2 and rat GLYT2. They show high homology(at least 40%) in their primary structures, withÇ150 well-channel openings whose probabilities depend on the

concentration of the substrate and co-ions (e.g., 1 open- conserved amino acid residues. As general features (seereferences in Table 4), all these transporters lack a signaling/350–700 transport cycles; Ref. 301). The channel

openings would account for the macroscopic current peptide, a good prognosis for 12 TM domains, with theNH2 and COOH termini located intracellularly, wherethat exceeds the charge flux expected from transport

flux and substrate/co-ion stoichiometry (301, 507). most putative phosphorylation sites are located, and oneto four putative N-glycosylation sites between the theoret-Transporters of this superfamily produce cation-perme-

able channel activity in the absence of substrate. This chan- ical TM domains III and IV. The regions of highest homol-ogy are TM domains I, II, and IV–VIII; in contrast, thenel mode of action is due to the expression of the corre-

sponding transporter, since it is absent in the nontransfected lowest level of homology occurs in the NH2 and COOHtermini (see Fig. 3 and Ref. 12 for review). Regions ofcells or in uninjected oocytes and shows the same pharma-

cological sensitivity as the transporters (507). Constitutive extensive homology may be related to general functionsfor these transporters. In general, there is good conserva-transporter leak currents (i.e., flux of driving ions down their

electrochemical gradient in the absence of substrate) have tion of proline, glycine, tyrosine, and tryptophan residuesin TM domains (see Fig. 3). Thus every theoretical TMbeen detected for the expressed human NET, rat SERT,

human DAT, and rat GAT-1 (81, 82, 161, 340, 520). Similarly, domain has at least one conserved glycine residue; prolineresidues are conserved in TM domains I, II, V–VIII, XI, andother sodium-dependent cotransporters show leak currents,

like the glutamate transporters EAAT1 and EAAT3 (see sect. XII; and tyrosine residues are conserved in TM domains I–III, V, and X. Transmembrane domains I and IV presentIIC), and the sodium/glucose cotransporter SGLT1 (reviewed

in Ref. 609). Interestingly, for many of these transporters one conserved positive amino acid residue. The structure-function relationship of some of these conserved struc-(rat GAT-1, rat SERT, and human DAT), the ion selectivity

of the leak conductance is similar but not identical to the tural features has been addressed experimentally (seesect. IIB5).cotransporter activity (alkali metal cations like sodium, po-

tassium, or lithium produce the leak conductance, whereas These transporters are N-glycoproteins. The GABAtransporter GAT-1 was reconstituted and purified to ho-potassium and lithium do not couple the transport of the

corresponding substrates; reviewed in Ref. 507). This raises mogeneity from rat brain (441, 439) and later cloned (184).The transporter was resolved in SDS-PAGE as a polypep-the question as to whether the leak conductance and the

translocation of the co-ions use the same permeation path- tide band of 80 kDa that also dimerizes to an apparentmolecular mass of 160 kDa, as revealed by cross-reactivityway. In any case, the leak pathway depends on the presence

of substrate: application of substrate inhibits this conduc- with polyclonal antibodies (439). The deglycosylatedGABA transporter has an electrophoretic mobility com-tance associated with the expressed rat GAT-1, rat SERT,

and human DAT (82, 340, 507). Patch-clamp studies by Cam- patible with a molecular mass of Ç67 kDa, deduced fromthe cloned GAT-1 transporter (184, 259). Other studiesmack and Schwartz (82) in HEK 293 cells, which perma-

nently express rat GAT-1, suggest that there are two distinct have also demonstrated that GAT-1 is N-glycosylated (37,273, 410, 439). Similarly, N-glycosylation has been demon-subpopulations of GAT-1 transporters: 1) the vast majority

of the transporters act as normal c-transporters that yield strated for DAT, SERT, NET, and GLYT1 transporters (70,307, 357, 358, 400, 468, 415, 547, 637).only small currents, and 2) a small ‘‘channel-like’’ population

produces leak conductance in bursts (Ç1 pA at 050 mV) Purification to apparent homogeneity of a pig brainglycine transporter showed it to be a glycoprotein (it bindsand is sensitive to the GABA GAT-1-specific inhibitor SKF-

89976A. For these authors, the large discrepancy between to lectins) with the appearance of a broad band with anaverage size of Ç100 kDa in SDS-PAGE (320). Physico-the number of channels and transporters indicates that they

function independently. The question is then, What mecha- chemical characterization of the native glycine trans-porter indicated a monomeric structure of this size (321).nisms determine whether a protein functions as a channel

or as a transporter (82)? Posttranslational modifications, the Treatment with peptide N-glycosidase F (PNGase F), butnot with endoglycosidase F or O-glycanase, produced astate of aggregation or interation with other proteins, like

cytoskeletal elements, or even silent channels, might be the dramatic electrophoretic mobility change, which showsthat Ç30% of the transporter mass corresponds to themolecular basis for the channel behavior. Purification and

reconstitution of these transporters are clearly needed to carbohydrate moiety; neuraminidase produces a slight re-duction of its apparent mass (395). The size of the degly-demonstrate that they could work intrinsically as channels.cosylated and glycosylated glycine transporter purifiedfrom pig brain suggests that it most probably corresponds4. Protein structureto the GLYT2 transporter (314, 395). Interaction with suc-cinylated wheat germ agglutinin lectin, but not with con-Figure 3 shows the alignment of the human amino

acid transporters of this superamily, with the exception canavalin A-lectin, and the previous glycosidase treat-


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ments indicate a tri- to tetra-antenary complex structure occurring N-glycosylation sites, whereas loop IL2 is notglycosylated; therefore, two TM domains should be placedwith terminal sialyc acid residues for the carbohydrate

moiety of the purified GLYT2 transporter (395). With a in between the TM domains III and III* shown in Figure4A. There is a hydrophobic segment of 44 amino acidsimilar approach, the carbohydrate moiety of the human

DAT transporter was shown to lack high-mannose resi- residues within these transporters that could accommo-date both TM domains (see Fig. 4A). In addition, studiesdues (306).

As indicated before, the cloning of the first two mem- with permeant and impermeant methanesulfonate re-agents indicate that the cysteine residue 74 (see Fig. 4A)bers of this superfamily, GAT-1 and NET, suggested a

common 12 TM domain model that was extended to the should be located intracellularly, as proposed by the newmodel, and not extracellularly, as proposed by the previ-other members of the family (see Fig. 3). This model ini-

tially received some experimental confirmation. Site-di- ous theoretical model; consistent with this, the hydro-philic loop containing cysteine-74 (i.e., the previous EL1)rected mutagenesis showed that the glycine transporter

GLYT1 is heavily glycosylated at four Asn residues within is not glycosylated (38). Aragon and Gimenez and co-workers (401) also used N-glycosylation scanning muta-the loop EL2 (see Figs. 3 and 4B), demonstrating the extra-

cellular location of this hydrophilic loop (400, 401). N- genesis to show that the loop connecting the TM domainsII and III can be glycosylated in vivo, and must thus beglycosylation within this loop has also been demonstrated

for SERT (547), NET (70, 358), and GAT-1 (38) transport- extracellular. These results suggest the model proposedin Figure 4B for rat GLYT1, where the TM domain III*ers. Immunofluorescence and electron microscopic stud-

ies in permeabilized and nonpermeabilized cells express- should contain a stop transfer signal. Unfortunately, thishas not been demonstrated in studies of fusion reportering GLYT1 and NET (623, 16, 70), and N-glycosylation

scanning mutagenesis for GAT-1 and GLYT1 (38, 401) con- glycosylation (401). This suggests that the proposed TMdomains III and III* constitute a single ‘‘reentrant loop’’firmed the intracellular location of the NH2 and COOH

terminals of these transporters. Similar evidence obtained as proposed for the first highly conserved hydrophobicdomain. In this sense, it is not easy to conceive two TMfrom experiments using antibodies located the hydro-

philic loop of human NET connecting TM domains VIII domains within the hydrophobic segment of 44 amino acidresidues as proposed by Bennet and Kanner (38) (see Fig.and IX intracellullarly (357), and those connecting TM

domains III and IV and VII and VIII (loops EL2 and EL4 4A). Indeed, no clear support for the intracellular locationof the hydrophilic loop IL2 has been offered; in fact, thein Fig. 4, A and B) extracellularly (70). N-glycosylation

scanning mutagenesis unambiguously showed the extra- construct of glycosylation reporter fusion of GLYT1 span-ning the NH2 terminus to the TM domain III is glycosylatedcellular location of loops EL3 and EL6 (see Fig. 4A) of

GAT-1 (38) and of loop EL3 of rat GLYT1 (401). N-glyco- in vitro (401).At present, the only evidence for the membrane inter-sylation of loops EL4 and EL5 (see Fig. 4B) has been

shown for rat GLYT1, but with an impaired transport func- action of the reentrant hydrophobic loop I (see Fig. 4A) isthat alkaline stripping does not release from microsomestion (401). For rat GLYT1, all this has been confirmed by

in vitro glycosylation reporter fusion; TM domains VI, VIII, a GLYT1 construct having only the COOH terminus, thereentrant domain I, and a small part of the new IL1 loopand X act as stop transfer signals for the preceeding hy-

drophobic TM domains V, VII, and IX, which act as mem- (401). Reentrant hydrophobic loops (or ‘‘pore loops’’) havebeen described for receptors and channels (39, 215, 362)brane anchor signals (401). The extracellular location of

the loop EL6 of rat GLYT1 is supported by similar experi- and are involved in ion permeation of voltage- or ligand-dependent ion channels (for review, see Ref. 333). Thisments showing that TM domain XII acts as a stop transfer

signal when placed after TM domain XI and the loop EL6 might help us to understand the channel mode of actiondescribed for several transporters within this superfamily.(401). In summary, the theoretical model of 12 TM do-

mains between the extracellular loop EL2 and the COOH In addition, several amino acid residues that are critical forthe transport function of the transporters of this superfam-terminus is supported by strong experimental evidence.

Very recently, two back-to-back reports examined in ily have been described within this hydrophobic domain(see sect. IIB5). It is therefore important to clarify, by differ-depth the membrane topology of GAT-1 (38) and GLYT1

(401) and by different strategies reached the same conclu- ent strategies, the membrane topology of these transporterswithin their NH2-terminal third.sion: the highly conserved theoretical TM domain I is

probably immersed in the plasma membrane, but doesnot span it completely. In addition, a new TM domain III* 5. Structure-function relationshipis proposed. This new 12 TM domain model is depictedin Figure 4, A and B. Bennet and Kanner (38) show that Site-directed mutagenesis with rat GAT-1 and GLYT1

transporters by Kanner’s, and Aragon’s and Gimenez’sthe previous intracellular loop connecting TM domains IIand III (i.e., new loop EL1, see Fig. 4A) can be glycosylated groups (16, 37, 50, 271, 288, 331, 410), a cross-mutation

study between the external loops of mouse GAT-1, -2,in vivo, as well as loop EL2, which contains the naturally


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and -3 and BGT-1 by Nelson and co-workers (542), exami- no rescue of transport activity when the transporter issolubilized and reconstituted from HeLa cells expressingnation of the role of the carbohydrate moiety for the gly-

cine transporters by Aragon and co-workers (395, 400), the mutants); only the deletion mutant of valine-348 showsa missorting defect, which is partially responsible for theand a substrate-protected proteolytic cleavage study of

rat GAT-1 by Mabjeesh and Kanner (332) are the bases loss of its transport activity. Interestingly, conserved andnonconserved (mutations to glycine) substitutions offor our present knowledge of the structure-function rela-

tionship of the sodium- and chloride-dependent amino these nonconserved residues within the superfamily oftransporters in rat GAT-1 retain significant transport activ-acid transporters. These data are discussed here in the

light of the new membrane topology model described for ity when expressed in HeLa cells (256). These results sug-gest that a minimal length of the hydrophilic loops EL4GAT-1 and GLYT1 transporters (see sect. IIA). These stud-

ies show that the NH2-terminal third of these proteins may and IL5 is important for the transporter functional struc-ture (256). In agreement with this, insertion of an Asnbe involved in common transport functions, whereas the

external hydrophilic loops affect substrate discrimination. residue between valine-348 and threonine-349 ensures sig-nificant transport activity (256).In a series of studies, Kanner’s group (37, 256, 331)

identified domains of rat GAT-1 that are not required for As discussed above (transport characteristics section),GAT-1 transports GABA, whereas GAT-2 and GAT-3 andits transport function. Papain- or pronase-treated purified

rat GABA transporter, which has probably lost the NH2 BGT-1 transport b-alanine in addition to GABA; all fourGABA transporters show different apparent affinity forand COOH termini, nevertheless retains all the transport

characteristics of the intact GAT-1 in a reconstituted sys- GABA (see Table 5), and there are specific amino acidresidues for the b-alanine transporters (i.e., GAT-2, GAT-tem (259, 331). In a parallel study, deletion mutants of

rat GAT-1, where most of the hydrophilic NH2 and COOH 3, BGT-1, and TAUT) between TM domains VII and VIII,IX and X, and XI and XII (see Fig. 3). Nelson and co-terminals have been eliminated, also retain all the transport

characteristics of the intact transporter when expressed in workers (542) examined the role of the external loops EL3to EL6 (see Fig. 4A) in substrate selectivity by cross-muta-HeLa cells (37). These results demonstrate that neither

terminus of the GABA transporter is needed for its trans- tion between the four GABA transporters (GAT-1 to-3 and BGT-1) (i.e., swapping amino acid residues betweenport function. These protein domains might be relevant for

other functions, like regulation of the transport function, these transporters) and expressing the mutants in oocytes(542). The most relevant results in this study are as follows.or interaction with cystoskeleton, as described for other

transporters (e.g., erythrocyte anion transporter, sodium/ 1) Expression of a mutant produced by swappingamino acid residues of the EL4 loop of GAT-3 (also veryproton exchanger; Refs. 290, 472). These results cannot be

generalized to other transporters of this superfamily. In similar to GAT-2 sequence) into GAT-1 sequence resultedin an apparent affinity change for GABA that mimics GAT-contrast to GAT-1, the COOH terminus of rat GLYT1 is

necessary for the correct trafficking of the protein to the 3 transport (Km values were Ç9, Ç1, and Ç2 mM formouse GAT-1, GAT-3, and the cross-mutant GAT-1/GAT-plasma membrane (399). Deletion of the first 30 amino acid

residues of GLYT1a (i.e., NH2 terminus) does not alter the 3-EL4 loop/GAT-1, respectively).2) The cross-mutant GAT-1/BGT-1-EL6/GAT-1 has in-transport of glycine by the transporter when it is expressed

in COS cells. Deletion of the last 34 amino acid residues creased affinity for GABA (Km Ç35 mM) that mimics BGT-1 transport (Km õ100 mM).(i.e., COOH terminus; see Fig. 4B) does not impair the

transport function of GLYT1a, but longer deletions within 3) The cross-mutant GAT-1/GAT-2-EL5/GAT-1 ac-quired similar b-alanine sensitivity to that of GAT-2, whichthe COOH terminus result in a progressive decrease of its

transport expression. Immunofluorescence and transport is not present in GAT-1. Cross-mutations within the otherloops do not confer b-alanine sensitivity, and swappingreconstitution studies showed that this impaired transport

expression was due to both a defect in trafficking to the amino acid residues of the EL5 loop of GAT-1 into theGAT-2 sequence reduced b-alanine sensitivity.plasma membrane and a loss of the intrinsic transport ac-

tivity of these mutants (399). 4) Cross-mutations within the EL3 loop produce nosignificant changes.Two studies analyzed the role of the hydrophilic

loops in the function of the GABA transporters (542, 256). Therefore, this elegant study strongly suggests thatthe three external loops EL4–6 (see Fig. 4A) might formKanner et al. (256) examined the effect of deletions of

randomly picked nonconserved segments, or even dele- a pocket on the transporters into which the substratesbind (542). Interestingly, a similar conclusion was reachedtion of single residues, within the EL4 and IL5 loops of rat

GAT-1 (single residues deleted are indicated by a square in in studies with chimeric biogenic monoamine transport-ers (71, 169). More recent studies (72) also showed thatFig. 4A). All deletions, of 1–22 residues, produce a loss

of transport activity. This could not be explained by a low the protein regions spanning TM domains V–VII and I–III of NTE and DAT are involved in the selective inhibitionlevel of synthesis (i.e., normal [35S]methionine labeling)

or by a missorting to the plasma membrane (i.e., there is by antidepressants and psychomotor stimulants.


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Four studies by Kanner’s group look for critical critical for the transport cycle-associated conformationalchanges of GAT-1 transporter (271).amino acid residues for the transport activity of rat GAT-

1, within charged residues (410) or conserved tryptophan Several lines of evidence highlight the relevance ofthe reentrant hydrophobic loop I and the adjacent proteinor tyrosine residues located in the theoretical TM domains

(50, 288), or conserved negatively charged residues adja- regions for the transporters of this superfamily (i.e., itmight be close to the permeation pore):cent to putative TM domains (271) (see Fig. 4A). Interest-

ingly, the three studies identified amino acid residues that 1) This domain shows high conservation within thetransporters of this superfamily and contains the criticalmay be critical for the transport function within the first

NH2-terminal part of the protein (see Fig. 4A), the mem- Trp-68 and Arg-69 residues.2) Deletion mutations of the NH2 terminus of rat GAT-brane topology of which has recently been revised (see

sect. IIB4). Of the five charged residues within theoretical 1 and GLYT1 affecting amino acid residues adjacent tothis domain abolish transport function (16, 37).TM domains, only Arg-69, located in the highly conserved

reentrant loop (38, 401), is critical for the radiolabeled 3) The cysteine residue 74, located in the adjacentIL1 loop (see Fig. 4A), is partially responsible for the inac-transport of GABA; the arginine residue itself is critical

and not merely the associated positive charge (410). The tivation of rat GAT-1 by (2-aminoethyl)methanethiosulfo-nate (38).loss of transport function when the Arg-69 mutants are

expressed in HeLa cells is due to intrinsic transport de- 4) N-glycosylation scanning mutagenesis within theextracellular loop EL1 (see Fig. 4A) resulted in almostfects and not to protein synthesis or missorting effects

(410). The role of Arg-69 is unknown, but its positive complete loss of transport function.5) The glutamate residue 101, conserved in all thecharge and the fact that it is conserved in all the transport-

ers of this superfamily prompted the authors to suggest transporters of this superfamily and located in this EL1loop, has been found to be essential for the transportthat it might be involved in the binding of chloride (410).

The lack of negatively charged residues conserved in function of GAT-1 (271).6) Moreover, it is noticeable that the biogenic aminethe theoretical TM domains of rat GAT-1 that would be

critical for the transport function, and therefore candi- transporters have a specific aspartate residue within thisdomain (Asp-79 in the human DAT sequence; the aminodates for the binding of sodium (410) fostered the study

of the role of tryptophan residues that might interact with acid transporters of this superfamily have a conservedglycine residue in this position; Gly-63 in rat GAT-1; seetheir P-electrons with this co-ion within the putative TM

domains (288). Of the 10 conserved tryptophan residues Fig. 4A), which is critical for the binding of the aminegroup by human DAT (286).within putative TM domains, only Trp-68, Trp-222 and Trp-

230 resulted in impaired transport function when mutated In section IIB4, the presence of reentrant hydropho-bic domains in channels was discussed. It is thus im-to serine or leucine residues; Trp-230 mutants showed

missorting, whereas mutations in the other two trypto- portant to determine whether mutations within these do-mains (e.g., Arg-69, Trp-68) affect the channel mode ofphan residues had intrinsic transport activity defects

(288). Interestingly, Trp-68, contiguous to the critical resi- action of these transporters. In other words, do Arg-69,Trp-68, or adjacent domain (e.g., Glu-101) mutants showdue Arg-69, is conserved in all the transporters of this

superfamily, and the GAT-1 transport activity tolerates the characteristic channel mode of activity of rat GAT-1?If, finally, transporters of this family have intrinsic chan-substitutions of Trp-68 to other aromatic residues. This

suggested that these two contiguous residues might inter- nel-like activity, strategies developed for the study of theconducting pathways of channels will be useful. As dis-act with the transport co-ions sodium and chloride (288).

Moreover, capacitive charge movement studies in oocytes cussed by Lester et al. (301), substitution mutants of criti-cal residues in or near the permeation pathway shouldsuggest that the Trp68Leu mutant ‘‘locks’’ sodium onto the

transporter by preventing (or slowing) the intracellular affect single-channel conductance, open-channel block-ade, or ion selectivity. Only upon purification and recon-release of the substrate (339). As in all types of site-di-

rected mutagenesis study, it is also possible that both stitution will it be possible to demonstrate the intrinsicchannel mode of action of these transporters. Until then,residues influence the transport function by affecting the

transporter structure. the residues that are critical for transport and channelactivities could be interpreted as being involved in theThe study of negatively charged residues adjacent to

TM domains identified Glu-101, within the newly proposed substrate-transporter conformational changes needed toactivate an extrinsic channel activity. The study of mu-loop EL1 (see Fig. 4A), as being critical for transport func-

tion (271). Replacement of Glu-101 by Asp reduced the tants able to dissociate between transport and channelfunctions may also reveal the mechanism of the channeltransport activity of GAT-1 by 99%. Substitutions of Glu-

101 do not show the sodium transient currents, which are mode of action of these transporters. Lester et al. (301)proposed a variation of the ‘‘alternating access model’’indicative of conformational changes during the transport

cycle. This has led to the proposal that this residue is to explain the putative intrinsic channel activity of these


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transporters: the transporter is alternatively gated at the porters,’’ occurs within the external loop EL2 (see Figs.3 and 4, A and B). Aragon and co-workers (395, 400)intracellular and the extracellular face during a transport

cycle, but with low probability, whereas during its channel analyzed the role of the carbohydrate component of thepig brain purified glycine transporter and the expressedactivity events both gates remain open, allowing a contin-

uous aqueous phase through the transporter. To explain rat GLYT1 glycine transporter. Treatment of the purifiedand reconstituted pig brain glycine transporter (GLYT2)why the ion selectivity does not always coincide between

the transport and the channel activities (either for the with PNGase F results in the loss of Ç30% of its apparentmass and a substantial decrease in its transport activitypresent transporters, see above and Ref. 507, or for the

chloride conductance of the sodium, potassium-glutamate (395). These results do not reveal whether the carbohy-drate moiety is important for substrate binding or for sta-transporters; see sect. IIC) the substrates and co-ions

themselves might become part of the ion-selective path- bilizing the active conformation of the transporter. Thisstudy cannot be extended to other transporters of thisway, as proposed for the glutamate transporters (582).

The critical residues Trp-222 and Trp-230 are located family. Indeed, N-glycosidase F treatment of the purifiedand reconstituted GLYT1 glycine transporter expressedwithin TM domain IV (see Fig. 4A). Tryptophan-222 is con-

served in the amino acid transporters, but not among the in COS cells results in the loss of its carbohydrate moietywithout loss of its transport function; N-glycosylationmonoamine transporters of this family (see Fig. 3 and Ref.

253). This fosters the hypothesis that Trp-222 might be does not appear to be essential for the transport activityof GLYT1 (400). On the other hand, the carbohydrate moi-involved in the binding of the amino group of these sub-

strates (288). Further support for this role of Trp-222 has ety of GLYT1 is indeed necessary for its proper traffickingto the plasma membrane. Progressive disruption of thebeen obtained by substitution mutants of Trp-222 that do

not bind radiolabeled tiagabine, a NIP derivative inhibitor four naturally occurring N-glycosylation sites of GLYT1in loop EL2 (see Fig. 4B) results in a progressive decreaseof GAT-1 that is not transported (253). As discussed above,

the biogenic amine transporters have a specific aspartate in the transport activity when the mutants are expressedin COS cells; surface biotinylation and immunofluores-residue within the reentrant hydrophobic loop that seems

to mediate the binding of the amine substrates (286). Of cence analysis demonstrated missorting to the plasmamembrane of the unglycosylated GLYT1 mutants (400).the 12 conserved tyrosine residues predicted to be located

in the membrane according to the theoretical topological This is consistent with the behavior of nonglycosylatedNET mutants, which upon expression in COS cells hadmodel (see Fig. 3), Tyr-140 (located in TM domain III or in

the intracellular loop IL2, depending on the topology reduced protein stability and surface trafficking, whichcould explain the loss of transport expression. Interest-model; see Figs. 3 and 4A) is the only one that does not

tolerate, in rat GAT-1, replacement by either phenylalanine ingly, the residual transport activity conserves substrateand antagonist recognition (358), and loop EL2 of SERTor tryptophan (50). A detailed study on substitution mu-

tants of residue Tyr-140 of rat GAT-1 expressed in HeLa (and probably NET) transporter does not appear to partic-ipate in substrate or antidepressant binding. Swappingcells or oocytes demonstrated that this residue is a specific

determinant on GABA binding (i.e., sodium and chloride half of this loop of NET into SERT transporter does notalter substrate or drug affinity but slows the transportbinding are unimpaired) (50). Interestingly, this tyrosine

residue is conserved throughout this superfamily of trans- rate; this suggests that loop EL2 may be involved in thetranslocation mechanism (511).porters (including the transporters for biogenic amines).

This allowed the authors (50) to suggest that Tyr-140 may The last item to be discussed on the structure-func-tion relationship of these transporters is the evidence inbe involved in the binding of the amino group, the moiety

which is common to all the substrates of this superfamily favor of transport cycle-associated conformationalchanges. An elegant study by Mabjeesh and Kanner (332)of transporters. Until the membrane topology of the trans-

porters of this superfamily is resolved (see sect. IIB4), it is provides experimental evidence of this for rat GAT-1transporter. The GABA transporter from brain mem-impossible to know which other group might be located

close to Tyr-140 (50), but the results discussed here (50, branes or purified and reconstituted into liposomes iscleaved by proteases (pronase and trypsin), and its trans-286, 288) suggest that this residue and Trp-222 (rat GAT-1

sequence) or Asp-79 (human DAT sequence) are determi- port activity is abolished. The presence of GABA, togetherwith the transport co-ions (sodium and chloride) on thenants of the binding of the amino group of GABA or bio-

genic amines, respectively. This suggests proximity for same side of the membrane, almost entirely blocked theaction of the proteases, both in the proteolysis of thethese residues within the three-dimensional structure of

these transporters. In any case, all these results confirm GAT-1 transporter and in its transport function (332). Theconcentration of GABA necessary for the half-maximalthe relevance of the NH2-terminal third of these transport-

ers for their transport function. protection, the partial protection by the GAT-1-specificinhibitor ACHC, and the lack of protection by b-alanineThe N-glycosylation of the transporters of this super-

family, with the possible exception of the ‘‘orphan trans- (noninteracting with GAT-1 transporter) (see Table 5) in


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addition suggest that the GAT-1 transporter undergoes transmission in the GABAergic synapses. The blockadeof GABAergic transmission precipitates epileptic seizuresconformational changes after binding of its substrate and

co-ions. Mabjeesh and Kanner (332) propose that the for- (for review, see Refs. 56, 157). Interestingly, lipophilicderivatives of piperidencarboxylic acid, which have anti-mation of the translocation complex (GABA plus co-ions)

induces conformational changes that render the trans- convulsant properties (524), are highly specific inhibitorsof GAT-1 transporter (58, 102, 103); most probably, GAT-porter resistant to digestion by these proteases. As for

other substrate-protection strategies, it is not possible to 1 transporter is the site of action of the anticonvulsantdrug tiabagine (56). The GAT-1 uptake system is moredistinguish between protection due to steric hindrance by

the substrate or conformational changes hiding protein extensive than the GABA synthesizing system, and it isalso expressed in glial cells of the cerebral and cerebellardomains. This discussion is relevant, since Lester and

co-workers (520) recently developed a new model of cortex and spinal cord (364, 375, 447, 506). Then, GAT-1should play additional roles to its presynaptic function.ion-coupled cotransport for GAT-1, SERT, and the so-

dium-glucose cotransporter (SGLT1) based on substrate- In this sense, it might contribute to the regulation of thecerebrospinal concentration of GABA. To our knowledge,substrate interactions that does not require the global con-

formational changes characteristic of the paradigmatic antisense or knockout experiments on GAT-1 to demon-strate this have not been reported.alternating access model. In any case, the study of the

capacitive properties of the GAT-1 transporter also pro- The channel mode of action of GAT-1 suggests a rolefor this transporter in intracellular signaling. Sonders andvides some clues to the conformational changes associ-

ated with the binding of sodium and GABA (see Ref. 301 Amara (507) have recently reviewed the potential physiolog-ical role of the GABA transporter-associated electric activ-for review). The slow component of capacitive currents

is associated with sodium binding, and it is compatible ity; at present, evidence in favor of this role is less docu-mented than that for the excitatory amino acid transportersboth with sodium ions entering the membrane dielectric

field and with reorientation of dipoles after sodium bind- (see sect. IIC). Application of GABA to glial cells eliciteddepolarization in a sodium-dependent manner, which ising (i.e., conformational changes) (301). The fast capaci-

tive currents due to stable expression of GAT-1 in HEK therefore more attributable to transporters than to recep-tors (291). Depolarization caused by the transport of GABA293 cells show an increased current noise in the mem-

brane patch after addition of GABA that increases with can trigger intracellular signals. In isolated skate retinalhorizontal cells, GABA-evoked transport currents (as deter-frequency (82), suggesting that GABA binding facilitates

sodium binding by eliminating barriers to the entry of mined by the pharmacology and ion dependence) depolar-ize cells and open voltage-sensitive Ca2/ channels (200).ions from solution (301). Similarly, jumps in the sodium

concentration produce charge movements that reflect the The pharmacological characteristics of the GAT-2and GAT-3 transporter fit the GABA glial transporter activ-same population of charges that move during voltage-

jump relaxations (339). All this suggests that the binding ity (see above), but GAT-3 is present in glial and neuronalcells (61, 103, 450). Studies in the developing brain (239)of GABA and sodium induces conformational changes in

the transporter (301). showed a coordinate expression of GAT-1 and GAT-3 thatsuggested a role for the latter in the termination of GABA-ergic synapses. In contrast, GABA uptake inhibitors with6. Physiological roleanticonvulsant properties do not inhibit uptake via GAT-3 (102). Nelson and co-workers (240) noticed that theA) TRANSPORTERS FOR NEUROTRANSMITTER AMINO ACIDS.

Sodium-dependent transporters located in the presynaptic general pattern of GAT-3 immunoreactivity is similar tothat of GLYT1, which suggested a possible role of GAT-3and glial membranes remove transmitter molecules (e.g.,

GABA, glycine, biogenic amines, and glutamate) from the in neurons with GABA termini known to be apposed toglycine receptors. As discussed above, the localization ofsynaptic cleft and thus terminate transmission. This has

been directly demonstrated for the dopamine transporter GAT-2 in brain, retina, and peripheral tissues points to anonneuronal function for this transporter.in the DAT knockout mouse; in the homozygotic mice,

dopamine persists Ç100 times longer in the extracellular There is codistribution in brain of the neuronal-spe-cific glycine transporter GLYT2, the inhibitory glycine re-space (168). g-Aminobutyric acid is the major CNS inhibi-

tory neurotransmitter, and glycine has this major role in ceptor (immunocytochemistry and strychnine binding),and neurons with high glycine content (17, 237, 623). Inspinal cord and brain stem. As discussed above, three

high-affinity GABA transporters are expressed in the CNS, contrast, codistribution of GLYT1 and inhibitory gly-cinergic neurotransmission is not complete (for review,GAT-1, -2, and -3. Biochemical, pharmacological, and im-

munolocalization studies demonstrated that GAT-1 corre- see Ref. 622). First, this suggests participation of bothglycine transporters in the termination of glycinergic syn-sponds to the neuronal subtype GABAA transporter (see

Ref. 260 for review). Therefore, GAT-1 should be resposi- apsis. Second, GLYT1 might have additional roles in theCNS. The presence of GLYT1 (mRNA and protein) in neu-ble for the presynaptic removal of GABA for terminating


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rons is controversial, but its presence in areas without implicated in retinitis pigmentosa, and depletion of tau-rine results in retina degeneration; this supports a role ofglycine receptor expression suggests additional roles

(622). Based on the mRNA distributions, Smith et al. (502) taurine in neuronal survival (316, 368, 503). Although mostanimals can synthesize taurine, this is not sufficient andpostulated a role for GLYT1 in the modulation of the gluta-

matergic transmission through NMDA receptors. This re- the supply relies on dietary sources (503). This role fortaurine transport is consistent with the presence of TAUTceptor requires two coagonists, glutamate and glycine, to

activate its channel (234, 287). Administration of glycine transporter in the blood retinal barrier (retinal pigmentepithelium) (368) and in the placenta (445). A more pre-or related agonits potentiates NMDA receptor function in

several models in vivo (reviewed in Ref. 622). The concen- cise cellular localization in the brain of the high-affinityTAUT transporter (see sect. IIB1) is needed to understandtration of glycine needed to restore normal NMDA electric

responses is controversial, and this is an important issue its role in the CNS.The cloning of PROT transporter, a sodium- and chlo-in assessing the role of the glycine transport in the modu-

lation of glutamatergic transmission; 0.1 mM glycine is ride-dependent high-affinity L-proline transporter ex-pressed in CNS, suggested a role for L-proline in neuro-enough to restore normal NMDA responses, but it is esti-

mated that glycine concentrations in the cerebrospinal transmission. Circumstantial evidence implicates L-pro-line as a putative synaptic regulatory molecule (see Refs.fluid are ú10 mM (622). Two findings leave room for a

role of glycine transport in NMDA receptor function. First, 155 and 489 for review).1) As for neurotransmitters, high-affinity sodium-de-the affinity of the NMDA receptor for glycine varies in

different neurons (432), depends on the type of NR2 sub- pendent uptake of L-proline has been described in ratbrain synaptosomes and slices, and L-proline is releasedunits within the receptor (584), and increases with extra-

cellular calcium (182). Second, the concentrative glycine from brain slices and synaptosomes by potassium-in-duced depolarization. In general, the pharmacology (po-transport present in brain synaptic plasma membrane and

glial cells, with a cotransport stoichiometry of 1 glycine, tency of inhibitors) of the L-proline transport of the humanPROT expressed in HeLa cells is consistent with the up-2 sodium, and 1 chloride (16, 624) (to our knowledge the

stoichiometry for the expressed GLYT1 transporter has take of L-proline in brain slices.2) L-Proline and its high-affinity synaptosomal trans-not been reported) is able to maintain a concentration of

0.2 mM in the synaptic cleft (23). The finding of brain port show heterogeneous regional distribution in the CNS.3) There is a synaptosomal L-proline biosyntheticregions with high NMDA receptor expression without gly-

cinergic terminals also points to a role of the glycine trans- pathway from ornithine.4) L-Proline produces complex electrophysiologicalporter in the regulation of glutamatergic synapsis. The

GLYT1 transporter might regulate glycine concentration actions when iontophoresed onto neurons.5) Intracerebral injections of L-proline are neurotoxicin the synaptic cleft, not only by the uphill uptake of

glycine, but also by the efflux of glycine via the transporter and disrupt memory processes. Interestingly, the neuro-toxic effects of L-proline are blocked by antagonists ofworking in the opposite direction (622). This is similar to

the nonvesicular release of GABA and glutamate (23). glutamate receptors.Transcripts of PROT are concentrated in subpopula-Thus, with synaptic depolarization, the intracellular con-

centration of sodium increases to levels at which the oper- tions of putative glutamatergic neurons, and the proteinis detected in synaptosomes (155, 489), and a specification of the glycine transporter is reversed (15). Addi-

tional studies are needed to demonstrate the role of the postsynaptic role of L-proline at glutamate receptors hasbeen suggested (for references, see Ref. 155). In this sce-GLYT1 transporter in changing the glycine concentration

in the synaptic cleft and thus modulating the NMDA gluta- nario, several roles have been proposed for the uptakeof L-proline via the neuronal PROT transporter (489): 1)mate receptor.

Transporters for two other amino acids, taurine and limitation, or modulation, of the extracellular concentra-tion of L-proline to prevent inadequate activation of, orproline, have been identified in brain. The physiological

role of taurine and b-alanine, two substrates of the TAUT modulate, inhibitory synapses (glutamate and glycine re-ceptor); and 2) a nutritional role for feeding the neuronaltransporter, in the CNS remains obscure. As reviewed

by Nelson and co-workers (316) and Weinshank and co- tricarboxylic acid cycle with intermediates or for presyn-aptic accumulation of L-proline as a precursor of L-gluta-workers (503), taurine is one of the most abundant amino

acids in brain, with a function that is best understood as mate. Recently, a novel nonopioid action of enkephalinshas been described (156): enkephalins, which are notan osmoregulator. Because taurine is degraded slowly,

uptake is a relevant way to regulate its extracellular con- PROT transporter substrates, competitively inhibit thistransporter. Subcellular localization, design of specific in-centration; taurine can be released from neurons and glial

cells in response to changes in cell volume. Taurine hibitors (156, 489), and knockout strategies for the PROTtransporter should help to elucidate the role of L-prolinereaches millimolar concentration in excitable tissues that

generate oxidants, a decrease in taurine uptake has been and its transporter in CNS.


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B) OSMOLYTE TRANSPORTERS. Both BGT-1 and TAUT BGT-1 gene. Electrophoretic mobility-shift assays withTonE-containing DNA and nuclear extracts from MDCKare established osmolyte transporters. Hypertonicity pro-

duces loss of cell water; cells shrink within seconds to cells revealed the presence of a specific binding protein(TonEBP) (538), whose binding activity is induced by hy-equalize intra- and extracellular osmolarity. An immediate

consequence, within minutes, is the elevation of the intra- pertonicity with a similar time course to the transcriptionof the BGT-1 gene (292). To our knowledge, the cloningcellular concentration of electrolytes (e.g., K/), which per-

turbs the function of macromolecules (for review, see of TonEBP has not been reported. Hypertonicity also re-sults in an increase in transcript abundance of TAUT andRefs. 193, 292, 293). Cells of all phyla have a fundamental

response, the accumulation of nonperturbing small or- SMIT (sodium/inositol) transporters (615, 562) and aldosereductase for the synthesis of sorbitol (499). A tonicity-ganic osmolytes (e.g., betaine, taurine, myo-inositol, and

sorbitol) (618). This response occurs in mammalian cells responsive sequence element has recently been identifiedin the mouse aldose reductase promoter, and it is similarwithin hours or days. Accumulation of compatible osmo-

lytes and keeping intracellular electrolyte levels isotonic to the TonE in the BGT-1 promoter (123). The cell tonicitysensor and its signal transduction pathway (e.g., involve-is critical for adaptation to hypertonic stress (for refer-

ences, see Ref. 292). The kidney medulla is the only mam- ment of mitogen-activated protein kinases) to TonE-de-pendent transcription in mammalian cells is not yet clearmalian tissue that is normally hypertonic, due to the con-

centration mechanisms of urine; depending on the hydra- (73, 294). Hormones, cumulative substrate uptake, andoxidative stress also produce the swelling/shrinking reac-tion status of the animal, osmolarity varies, and in

humans, it easily surpasses 1,000 mosM (193, 292). As part tion in a variety of mammalian cells; in addition to renalmedulla, osmolytes have been identified in astrocytes, he-of the renal mechanisms for water conservation, cells in

the medula accumulate betaine, myo-inositol, taurine, and patocytes, lens epithelia, and macrophages (for a shortreview, see Ref. 594). In mouse macrophages (RAW 264.7sorbitol; induction of transport for the first three osmo-

lytes and synthesis of sorbitol are the mechanisms that cells), as in MDCK cells, BGT-1 transcript abundance andbetaine uptake is strongly dependent on extracellular os-produce osmolyte accumulation (386, 387).

In a series of elegant studies, Handler’s and Burg’s molarity (593). In contrast, in H4IIE rat hepatoma cellsand primary hepatocytes, taurine is the most prominentgroups demonstrated the role and the mechanisms of reg-

ulation of BGT-1 transport activity in the adaptation of osmolyte, and its TAUT transporter is strongly regulatedby tonicity (594). To our knowledge, whether the L-prolinerenal cells to osmolarity changes (reviewed in Refs. 194,

292, 293). (PROT) and glycine (GLYT1) transporters, homologousto BGT-1 and TAUT transporters, are regulated by tonicity1) Hypertonic media induce betaine accumulation in

the canine renal MDCK cells (385). has not been reported.2) The MDCK cells rely on transport as a source of

betaine; the transport activity of BGT-1 is the rate-limitingC. Superfamily of Sodium-Dependent Transportersstep in the accumulation of betaine (387, 614).

for Anionic and Zwitterionic Amino Acids3) Addition of betaine at physiological concentration(100 mM) restores the MDCK cell growth and survivalchallenged by hypertonicity (563). This family of transporters comprises five anionic

amino acid transporters (EAAT1 to EAAT5; for excitatory4) In MDCK cells, hypertonicity induces the paralleland progressive turn-on of BGT-1 transcription, which raises amino acid transporter isoforms 1 to 5) and three zwitter-

ionic amino acid transporters (ASCT1, ASCT2, and ATBo;BGT-1 transcripts and transport activity (Ç6-fold increase);hypotonicity reverses this process (387, 564, 614). for system ASC transporter isoforms 1 and 2, and for

amino acid transporter for system Bo) that are sodium5) The BGT-1 transporter seems to play the same rolein renal medulla: renal preparations show sodium- and dependent (18, 144, 245, 269, 424, 488, 518, 568). The hu-

man homologs for all these transporters appear to havechloride-dependent betaine uptake (292), the increase inmedular tonicity due to dehydration results in an increase been cloned (18–20, 144, 249, 268, 343, 488, 490, 491, 568).

Indeed, most probably ASCT2 is the murine counterpart ofin BGT-1 transcript levels (quoted in Ref. 292), and intra-peritoneal administration of NaCl in rats rapidly increases human ATBo (see below). The cDNA accession numbers,

chromosome location, and protein length for the humanBGT-1 transcript abundance in renal medulla, mainly inthe thick ascending limbs of Henle’s loop (365). In these transporters are shown in Table 6. The simultaneous clon-

ing from different labs resulted in a confusing array ofwhole animal models, the expected increase in transcrip-tional activity of BGT-1 gene has not been reported. clone names (see Table 6). For clarity, in this review,

the nomenclature used by Amara’s group (11) has beenAfter cloning the complete BGT-1 gene (537),Handler and co-workers (538) identified the first-known adopted for the glutamate transporters (i.e., EAAT). On

the basis of sequence homology, two subfamilies couldtonicity-responsive sequence element (TonE; TGCAAA-AGTCCAG) 50–62 bp upstream of the first exon of the be interpreted: 1) the human EAAT isoforms (the anionic


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TABLE 6. Sodium-dependent transporters for anionic and zwitterionic amino acids

ProteinAccession Numbers Origin of First Clone and Human Amino Acid Clones in Other

Transporter (Gene Name) (Origin of Human Clones) Other Names Chromosome Length Mammals

Glutamate transportersEAAT1 (SLC1A3) L19158 (cerebellum) (490) Rat brain (GLAST) 5p13 (191, 284, 542 Bovine (225)

D26443 (brain) (268) (518) 535) Murine (191)U03504 (motor cortex) (19) GluT-1 (268) 5p11-p12 (516)

EAAT2 (SLC1A2) Z32517 (brain) (343) Rat brain (GLT-1) 11p12-p13 (310) 574 Murine (285, 382,U03505 (motor cortex) (19) (424) 11p11.2-p13 (534) 523)

GLTR (343)EAAT3 (SLC1A1) U06469 (kidney and ileum) (249) Rabbit small intestine 9p24 (500) 524 Rat (51, 581)

U03506 (motor cortex) (19) (EAAC1) (245) Murine (382)U08989 (brain stem) (490) Glutamate

transporter III(490)

EAAT4 (SLC1A6) U18244 (cerebellum) (144) Human cerebellum ? 564(144)

EAAT5 U76362 (retina) (18) EAAT5 (18) ? 560Zwitterionic amino

acid transportersASCT1 (SLC1A4) L14595 (motor cortex) (20) Human motor cortex 2p13-p15 (214) 532

L19444 (hippocampus) (488) (20)Human hippocampus

(SATT) (488)mASCT2* D85044 (mouse testis) (568) Mouse adipocyte ? 553

(AAAT) (311)ATB0 (SLC1A5) U53347 (JAR cells) (269) Human 19q13.3 (236, 269) 541

choriocarcinoma(269)

Pancreatic islet (236)cell lines

Accession numbers for human cDNA are indicated, except for ASCT2. Sequence homology, tissue distribution, mRNA size, and characteristicsof associated amino acid transport activity suggest that human ATBo and mouse ASCT2 may be species counterparts of same transporter.* Mouse ASCT2; human not available.

transporters) show 36–65% amino acid sequence identity tution showed L-glutamate transport activity; they raisedantibodies against the protein (121) and used them tobetween them, and 2) the human zwitterionic amino acid

transporters ASCT1 and ATBo show 57% between them. isolate GLT-1 cDNA (EAAT2 in Table 6). Transfectionof EAAT2 in HeLa cells resulted in a sodium-dependentAmino acid sequence identity between human ATBo and

mouse ASCT2 is very high (Ç80%). Amino acid sequence glutamate uptake that was thereafter reconstituted inliposomes (424). Finally, Hediger’s lab (245) isolatedidentity between members of the two subfamilies ranges

between 39 and 44%. Homologous prokaryotic proteins EAAC1 cDNA (EAAT3 in Table 6) from rabbit small in-testine by expression cloning in oocytes, after a sodium-(26–32% identity) involved in the transport of glutamate

and other dicarboxylates (140, 233, 558, 559, 586) are evo- dependent L-glutamate uptake expression signal. Theother transporters of this superfamily were cloned basedlutively related to this amino acid transporter superfamily

(205, 247, 252). on homology with these seminal sequences. The trans-port properties of EAAT1, -2, and -3 did not fulfill allThree independent labs cloned the first three mem-

bers of this superfamily almost simultaneously in 1992, the pharmacologically distinguishable glutamate uptakeactivities in the cerebellum and retina (119, 152, 454).using different approaches. Stoffel and co-workers

(518), during the isolation of a galactosyltransferase This was pursued by Amara’s group to isolate EAAT4cDNA from human cerebellum by using degenerate oli-from rat brain, purified a 66-kDa hydrophobic glycopro-

tein which upon protein microsequencing and oligonu- gonucleotides from two conserved regions of EAAT1–3 (i.e., one between TM domains III and IV, the other incleotide probe cloning resulted in the isolation of GLAST

cDNA (EAAT1 in Table 6); the putative protein showed the hydrophobic long stretch toward the COOH termi-nus of the proteins; see Fig. 5) as the starting point ofsignificant homology with prokaryotic glutamate and di-

carboxylate transporters, and its expression in oocytes a RT-PCR-based cloning strategy. Expression in oocytesconfirmed the sodium-dependent L-glutamate transportresulted in anionic amino acid transport activity. Kanner

and co-workers (120) purified to apparent homogeneity activity of EAAT4 (144). More recently, the same groupisolated EAAT5 from human retina (18), based on a pre-a rat glial 70- to 80-kDa glycoprotein that upon reconsti-


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vious glutamate transporter cDNA isolated from sala- 1) As a general rule, all these transporters show po-tassium dependence in addition to sodium dependencemander retina by homology strategies (not reported).

Independently, two groups cloned human ASCT1, the when expressed in different cell system, with the possibleexception of the zwitterionic amino acid transportersfirst zwitterionic amino acid transporter of the family

(also named SATT; see Table 6). Amara and co-workers ASCT1 (627), ASCT2 (568), and ATBo (269) (see Table 7).The characteristics of their expressed transport activity(20), using a degenerated oligonucleotide from a con-

served region of the long hydrophobic stretch of suggest that EAAT1–5 isoforms are neural and nonneuralvariants of the anionic amino acid transport system X0

AG,EAAT1–3 (see Fig. 5), isolated ASCT1 from motor cor-tex, and Fremeau et al. (155) isolated SATT from hippo- and that ASCT1 and ASCT2 might be variants of system

ASC transport activity, or that ATBo and ASCT2 corre-campus taking advantage of a human expressed se-quence tag (EST) (1) that showed slight homology with spond to the epithelial system system Bo.

2) Several labs provide evidence that some of thesean Escherichia coli glutamate/aspartate transporter(586). Both proteins upon expression in HeLa cells or transporters (EAAT1–5, ASCT-1, and perhaps ATBo) have

a substrate (amino acid and sodium)-gated chloride chan-oocytes exhibit sodium-dependent zwitterionic aminoacid transport activity with characteristics of system nel mode of action in addition to their amino acid trans-

port mode of action (18, 144, 270, 582, 583, 627; reviewedASC (20, 488). After the corresponding corrections, se-quences ASCT1 (accession no. L14595) and SATT (ac- in Ref. 507).

3) The topology of all these transporters in thecession no. L19444) are identical. Serendipity againadded to our structural knowledge of amino acid trans- plasma membrane is difficult to imagine on the basis of

hydrophobicity algorithms (6–10 TM domains have beenport. While screening a 3T3-L1 adipocyte cDNA libraryfor clones encoding protein tyrosine phosphatase HA2, suggested, including b-sheets TM domains; Refs. 245, 424,

518), and it is still not evident after experimental work,Liao and Lane (311) isolated a cDNA (AAAT in Table6) that showed significant homology with the previous from three different labs, attempting to elucidate this is-

sue for EAAT1, EAAT2, and a prokaryotic glutamate trans-members of this family of amino acid transporters. Inde-pendently, Kanai’s group (144), after a strategy based on porter (140, 498, 585).

4) Specific antisense experiments, both in vivo andRT-PCR amplification using degenerate oligonucleotidefrom similar conserved regions to those of Amara’s in cell culture, represent the first experimental evidence

of the physiological role of EAAT isoforms in brain (458).group (20), isolated ASCT2 cDNA from mouse testis(568). Expression in oocytes and transfection in 3T3-L1 More recently, Stoffel and co-workers (416) obtained the

null knockout EAAT3 mice, and Tanaka and co-workerspreadipocytes of ASCT2 and AAAT resulted mainly insodium-dependent zwitterionic amino acid uptake, also (544) obtained the null knockout EAAT2 mice.

These aspects as well as structure-function relation-with characteristics of system ASC (311, 568). Finally,a low-stringency screening of a human placental chorio- ship studies on these transporters are discussed in the

following sections.carcinoma cell cDNA library with a human ASCT1 probe(20) allowed Ganapathy and co-workers (269) to isolateATBo cDNA. Expression in HeLa cells and oocytes 1. Tissue expressionshowed a sodium-dependent uptake of zwitterionicamino acids with characteristics of system Bo (269). This The tissue distribution of the transporters of this su-

perfamily was studied initially by Northern analysis andgroup screened a mouse kidney cDNA library using ratATBo cDNA as a probe, and all the positive clones ob- in situ hybridization. Table 7 shows the mRNA tissue dis-

tribution and the mRNA size for these human transport-tained turned out to be ASCT2 (V. Ganapathy, personalcommunication). This, and the homology and the simi- ers, except for the mouse ASCT2. Rat EAAT1–2 and rabbit

EAAT3 show a similar tissue distribution and transcriptlarities of the amino acid transport associated with theexpression of ASCT2 and ATBo (see below) strongly sug- size (245, 424, 518); for the rabbit EAAT3, an additional

transcript band of 2.5 kb has been reported (245). For thegest that both cDNA are species counterparts of thesame gene. A search in dbEST performed by the authors human ASCT1, Shafqat et al. (488) reported two hybridiza-

tion bands of Ç2 kb (2.8 and 2.2 kb) instead of a singleof the present review (December 1996) found no otherEST clones indicative of new members of this superfam- band of Ç2.5 kb (20).

The human EAAT glutamate transporters have spe-ily of transporters in mammals.Since the identification of the first members of this cific tissue distribution. The EAAT2 isoform is specific to

the CNS, EAAT5 is mainly, if not solely, expressed intransporter superfamily, several excellent reviews haveappeared related to these glutamate transporters (205, the retina, and EAAT4 is mainly expressed in cerebellum,

whereas the other isoforms are also expressed in periph-243, 247, 252, 253). The study of the transporters ofthis superfamily has revealed intringuing concepts, as eral tissues (Table 7). Rat EAAT1 is also brain specific (see

below). Tissue and subcellular distribution of EAAT1–3follows.


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FIG. 5. Amino acid sequence comparison of excitatory amino acid transporter (EAAT) and related zwitterionicamino acid transporters. Alignment of amino acid sequence of 7 human transporters of this superfamily correspondsto that published by Arriza et al. (18) for EAAT1–5 isoforms. Amino acid residues present in at least 6 of listed sequencesare indicated by gray boxes. Amino acid residues that are specific to glutamate transporters or zwitterionic amino acidtransporters of this superfamily are in white on black boxes. Because of weak homology present in NH2 and COOHtermini of these transporters, conserved amino acid residues in those domains are not indicated. Straight lines oversequences indicate putative TM domains (I–VI). Dashed line over sequence delimits highly conserved, long hydrophobicstretch of amino acid residues with a controversial topology. Between TM III and TM IV, 2 or 3 potential N-glycosylationsites are conserved (open boxes). Dashes indicate gaps for sequence alignment.

/ 9j0c$$oc03 10-05-98 10:11:42 pra APS-Phys Rev

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TABLE 7. Tissue distribution and transport characteristics of expressed EAAT and related zwitterionic

amino acid transporters

Ligand-GatedTissue Distribution Expression Cotransported Chloride

Transporter (Transcript Size) System Substrates (Km) Pharmacology (Ki) Ligands Ion Leak Channel

EAAT1 Brain (other tissues ?)a Oocytese L-Glu, L-Asp, D-Asp SOS (Ç100 mM) aa0, (3) Na/ Yes (246, Yes (?) (582)(Ç4 kb) COS-7 cells (15–20 mM) DMG (Ç120 mM) (in) 579) (Erev /9

(19) PDC (Ç30 mM) LaAA (ú1 mM) K/ (out) (289) (Na/) mV)KA (ú3 mM) Electrogenic

(19, 289)EAAT2 Brainb (Ç10 kb) Oocytesf L-Glu, L-Asp, D-Asp KA (õ60 mM) Na/ (in), K/ No (579, Yes (582, 583)

COS-7 cells (10–20 mM) DMG (Ç0.7 mM) (out)i 583) (Erev /60(19, 285) PDC (õ10 mM) SOS (Ç1.2 mM) Electrogenic mV)

HeLa cellsg LaAA (ú1 mM) (19)EAAT3 Epithelia, brain, and Oocytesh L-Glu, L-Asp, D- SOS (Ç150 mM) aa0, (2/3)† Yes Yes (?) (582)

other tissuesc (Ç3.5 COS-7 cells Asp* (30–50 DMG (Ç0.25 Na/ (in) (246) (Erev /38kb) (19) mM) mM) K/, OH0 (out) mV)

PDC (Ç20 mM) LaAA (ú1 mM) (246, 628)KA (ú3 mM) Electrogenic

(19, 246)EAAT4 Brain and other Oocytes L-Glu, L-Asp, D-Asp KA (ú5 mM) Na/ (in) (144) (?) Yes (144)

tissuesd (Ç2.4 kb) (144) (2–3 mM) K/ (?) (Erev 022PDC (Ç3 mM) Electrogenic mV)La-aa (Ç200 mM) (144)

EAAT5 Retina (Ç3.5 kb) Oocytes L-Glu, L-Asp, D-Asp PDC (6 mM) Na/ (in), K/ Yes (?) Yes (18) (Erev

(other tissues ?) (18) (18) (13–64 mM) THA (1 mM) (?) (18) (18) 020 mV)ASCT1 Ubiquitous ? (20, 488) Oocytes L-Cys (30 mM), MeAIB (ú10 Na/ (in) (20, (?) Yes (627)

(°5, °4, Ç2.5 kb) (20) L-Ala mM) 488) (Erev 021HeLa cells L-Ser, L-Thr, L-Val K/ (?) (20, mV)

(488, 541) L-Glu 627)Electrogenic

(20)ASCT2 Several tissues (311, Oocytes L-Ala, L-Ser, L-Cys, MeAIB (ú1 mM) Na/ (in), K/ (?) No (?) (568)

(mouse) 568) (no brain or (568) and L-Gln (20 aa/ (ú1 mM) (?) (568)liver) (Ç2.7 kb) 3T3-L1 cells mM) aa0 (Ç1 mM) Electroneutral

(311) Branched aao (568)L-Glu (1.6 mM)

ATBo Epithelia and skeletal Oocytes and aao MeAIB (ú5 mM) Na/ (in), K/ (?) Yes (?) (270)muscle (269) (no HeLa aa/ (ú5 mM) (?) (269) (Erev 030brain or liver) (Ç2.9 cells (269) aa0 (ú5 mM) Electrogenic mV)kb) (269)

Transcript size and tissue distribution correspond to human transporters, except for ASCT2. Apparent Km values were obtained by differentlabs expressing corresponding transporters in Xenopus oocytes. Pharmacological characteristics of EAAT transporters and all the ligand channelcharacteristics correspond to those of human counterparts. * Apparent Km is dependent on membrane voltage (see text). † Two differentreports (246, 628) showed different stoichiometry for EAAT3 transporter. Question marks denote either inconsistent data or not tested (see textfor details). SOS, L-serine-O-sulfate; DMG, trans-(dicarboxyl)-2,4-methanoglutamic acid; LaAA, L-a-aminoadipate; KA, kainic acid; PDC, L-trans-pyrrolidine-2,4-dicarboxylic acid; THA, threo-b-hydroxyasparate; aa/, dibasic amino acid; aa0, dicarboxylic amino acid; aao, zwitterionic aminoacid. References are as follows: a) 19, 268, 491, 518; b) 19, 343, 382, 424, 523; c) 19, 245, 249, 382, 490; d) 144; e) 19, 115, 116, 225, 268, 289, 518,543, 571, 582; f) 19, 571, 582, 583; g) 83, 424, 426, 523, 630; h) 19, 245, 246, 249, 581, 582; i) 19, 424, 583. Other references are given in parentheses.

transporters in the CNS has been studied at the protein apses, suggesting a role in synaptic plasticity (461). Incontrast, glial EAAT2 localized to cell bodies and pro-level (85, 121, 298, 305, 461), largely confirming previous

in situ hybridization analyses (205, 245). These studies cesses (461). Neural EAAT3 localized to axons, dendrites,and presynaptic terminals in the deep cerebellar nuclei,showed that rat EAAT1 and EAAT2 are present in astro-

glial cells, whereas EAAT3 is present in neurons. Glial but in the cerebellar granule cells it is not expressed pre-synaptically (461). Astrocyte membranes facing nerve ter-EAAT2 is distributed throughout the brain and spinal cord

(most abundant in Bergmann glia in the cerebellar molec- minals, axons, and spines are higher in EAAT1–2 gluta-mate transporters than those facing capillaries, pia, orular layer); neuronal EAAT3 is also generalized, but it is

more prominent in hippocampus than cortex and striatum white matter (85). Interestingly, EAAT3 protein is presentin, but not restricted to, glutamatergic neurons (e.g., someand less abundant in cerebellum; and glial EAAT1 is most

prominent in cerebellum (cerebellum úú hippocam- but not all cortical pyramidal neurons), and it is enrichedpostsynaptically within GABAergic Purkinje cells in cere-pusú cortex ú striatum) (85, 298, 461, 581). Ultrastruc-

tural analysis revealed that glial EAAT1 is localized to bellum (461). This was confirmed by in situ hybridizationstudies (205, 581), where an additional localization ofprocesses that most probably envelop glutamatergic syn-


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EAAT3 to cholinergic a-motor neurons of the spinal cord placenta is very low. If this low level of expression alsooccurs at the protein level, to our knowledge not yet stud-was shown. These results indicate that neuronal EAAT3

is not the presynaptic glutamate transporter for all gluta- ied, different ASCT variants or transporters not related tothis superfamily may be responsible for the high systemmatergic synapses and that it is also present in nongluta-

matergic neurons. Additional glutamate transporters may ASC transport activity present in these tissues (488) (seesect. IIC8). The human ATBo transcript is present in epi-be present presynaptically in many glutamatergic neu-

rons. In this sense, the EAAT4 isoform may be a good thelial tissues (placenta, lung ú kidney, pancreas), veryscarce in skeletal muscle, and absent, according to North-candidate for those neurons in cerebellum. The presence

of neuronal EAAT3 in GABAergic synapsis suggests that ern analysis, in heart, brain, and liver. It has been pro-posed that ATBo corresponds to the apical (i.e., brush-it could transport glutamate intracellularly as a precursor

for GABA synthesis (461). The general role of EAAT1–3 border membrane) system Bo (Ref. 269; see below) andis therefore expected to be expressed in small intestine,isoforms in brain in the maintenance of low extracellular

glutamate concentration has been addressed by partial where this transport activity has been shown (329, 337,515). Indeed, rabbit ATBo cDNA has been isolated fromand specific knockout of these isoforms (458) and the

complete knockout of EAAT3 (416) and EAAT2 (544) (see jejunum (270). It is worth mentioning that the tissue distri-bution of ATBo is similar to that of ASCT2, again favoringsect. IIC8). To our knowledge, human EAAT4 and EAAT5

distribution has been studied only by Northern analysis, the hypothesis that both cDNA are species counterpart ofthe same gene. Immunolocalization studies to demon-showing an almost specific cerebellar and retinal localiza-

tion, respectively (144). It is worth mentioning that the strate the presence of ATBo in epithelial brush-bordermembranes are not yet reported.human EAAT5 has a putative synaptic localization: the

COOH terminus of EAAT5 contains a sequence motif (Glu-Ser or Thr-X-Val-COOH) found in synaptic membrane pro- 2. Transport propertiesteins and that interacts in the yeast two-hybrid assay toPDZ (a modular protein-binding motif) domains of the The transport properties of the glutamate (EAAT1–

5) and zwitterionic amino acid transporters (ASCT1–2postsynaptic density-95 protein (18).The peripheral distribution of EAAT1 (brain ú and ATBo) have been studied upon expression in oocytes

or transfection into mammalian cells followed by bothheartú skeletal muscle ú placenta and lung in humantissues, Ref. 19; specifically expressed in brain for the rat, radioisotopic and electric measurements (see Table 7).

Reconstitution studies from the expressed protein haveRef. 518), EAAT3 (small intestine ú kidney ú brain úcerebellum, lung, placenta, heart; similar for human and been reported only for the glutamate transporter EAAT2

(424, 426, 630). All these transporters share several trans-rat tissues, Refs. 19, 245), EAAT4 (cerebellum ú ú pla-centa, for human tissues, Ref. 144) and EAAT5 (weak port properties (see Table 7). 1) The glutamate EAAT

transporters are sodium cotransporters and potassiumsignals in liver and of different transcript size in skeletalmuscle and heart from human tissues, Ref. 18) isoforms countertransporters. Potassium dependence (counter-

transport) has been demonstrated for all of these trans-has been, to our knowledge, only addressed by Northernanalysis. It is noticeable that rat EAAT1 seems to be spe- porters, except EAAT4 and EAAT5, for which it has not

been reported (18, 144). 2) The zwitterionic amino acidcific to brain (518), whereas peripheral human tissuesexpress this isoform (19). Kanai and Hediger (245) per- transporters (ASCT1, ASCT2, and ATBo) are probably

electroneutral sodium-dependent amino acid exchangersformed in situ hybridization studies with EAAT3 in thesmall intestine and showed that the transporter is ex- that do not interact with potassium (162, 627), but to our

knowledge, the putative exchange mechanism of trans-pressed in epithelial cells. This, together with its expres-sion in kidney, suggests a participation of this glutamate port for ASCT2 and a role of potassium for ATBo and

ASCT2 has not been properly tested. 3) In addition totransporter in the reabsorption of anionic amino acids(see sect. IIC8). their transport mode of action, for EAAT1–5 and ASCT1

cumulative evidence has been obtained that they alsoThe tissue distribution of the zwitterionic amino acidtransporters ASCT1, ASCT2, and ATBo has been examined have a chloride channel mode of action (144, 582, 583,

627). Very recent data also suggest that this channel activ-by Northern blot analysis (20, 269, 488, 568). HumanASCT1 transcripts (Ç5, Ç4, and Ç2.4 kb; Ref. 20; and ity might be associated with ATBo (270). To our knowl-

edge, this has not been reported or tested for ASCT2,Ç4.8, Ç3.5, Ç2.8, and 2.2 kb; Ref. 488) were detected inskeletal muscle, pancreas ú brain ú placenta ú heart, and the only available study shows no electric activity

associated with this transporter (568) (see below).lung, kidney, liver. The mouse ASCT2 transcript (Table 7)shows a complementary tissue distribution: lung, large Substrate specificity has been described for the EAAT

isoforms and the zwitterionic amino acid transporters ofintestine, kidney ú skeletal muscle, testis and white adi-pose tissue (568). Expression of the two suspected trans- this superfamily (see Table 7). In constrast to these zwit-

terionic amino acid transporters, EAAT transporters ex-porter variants of system ASC (ASCT1–2) in liver and


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press high-affinity sodium- and potassium-dependent apparent Km values in those studies. It is worth men-tioning that for human EAAT3, dependence of the appar-transport of L-glutamate (micromolar range). In contrast,

the interaction of these transporters with zwitterionic ent Km on membrane potential has been described (249).Pharmacological studies helped to distinguish be-amino acids (i.e., either as a substrate or as a cis-inhibitor)

is of low affinity (millimolar range). All this information tween the transport activity of the five isoforms (see Table7). Sequence comparisons strongly indicate that humanis consistent with a large body of accumulated knowledge

on the native glutamate transport properties in cells, mem- EAAT1–3 corresponds to the rat GLAST1 (96% amino acidsequence identity; Ref. 518), rat GLT1 (95% amino acidbrane preparations, synaptosomes, and purified transport-

ers of neural origin (for review, see Refs. 251, 394). Inter- sequence identity to the corrected sequence; Refs. 253and 424), and rabbit EAAC1 (92% amino acid sequenceestingly, these EAAT isoforms do not interact with neuro-

transmitters that are substrates of the superfamily of identity; Refs. 245, 249). Because of this, in the presentreview, they are considered to be counterparts of the glu-sodium- and chloride-dependent transporters of neuro-

transmitters (e.g., GABA, dopamine, norepinephrine, sero- tamate transporter isoforms in different species. In con-trast, pharmacological similarities between species aretonin) (reviewed in Ref. 252). The five glutamate trans-

porter isoforms are stereospecific for glutamate but do less clear. Interaction of different inhibitors with thetransport activity of the human EAAT isoforms is shownnot discriminate the enantiomers of aspartate, a charac-

teristic of sodium-dependent glutamate transport activi- in Table 7. Human EAAT1 and EAAT3 show a similarpharmacology, which is different from that of humanties in many tissues (185, 262, 483, 505, 513); only for

human EAAT5 is the apparent Km for L-aspartate approxi- EAAT2 (19), EAAT4 (144), and EAAT5 (18). HumanEAAT2 is the isoform sensitive to dihydrokainic acidmately fivefold lower than that for D-aspartate (18). The

five glutamate transporters form a continuum of transport (DHK) and kainic acid (KA), insensitive to L-a-aminoadi-pate (LaAA), and relatively insensitive to L-serine-O-sul-activities with a different tissue-specific distribution, and

they are difficult to distinguish with the usual functional fate (SOS); human EAAT1 and EAAT3 are highly sensitiveto SOS and insensitive (i.e., Ki ú3 mM) to DHK, KA, andcriteria, other than by their pharmacology (see below).

Their activity corresponds to system X0AG, which repre- LaAA; and human EAAT4 is the isoform that is sensitive

to LaAA and insensitive to DHK and KA (Ki ú3 mM). Insents various transport agencies that carry anionic aminoacids, with the described characteristics, into different contrast to the other EAAT isoforms, for which THA and

PDC are competitive substrates, for human EAAT5 thesecells (41). Moreover, as expected for system X0AG (41),

threo-3-hydroxy-DL-aspartate (THA) is a competitive sub- analogs act as blockers (Ki Ç1–6 mM) and not as sub-strates (18). In constrast to the human EAAT transporters,strate of EAAT1–3 (19, 518). When the five human EAAT

isoforms are expressed in oocytes, the highest affinity is rat GLT-1 (rat EAAT2 counterpart) and rabbit EAAC1(rabbit EAAT3 counterpart) are very sensitive to LaAAshown by EAAT4 [apparent Km of 2–3 mM for L-glutamate,

L- and D-asparate, and the analog L-trans-pyrrolidine-2,4- (Ki in the micromolar range) (245, 424). These resultssuggest pharmacological differences between the humandicarboxylic acid (PDC)]; the other isoforms showed ap-

parent Km values for glutamate in the range between 10 and other EAAT isoforms. Brain regional differences inpharmacology have suggested at least four glutamateand 64 mM (18, 19). These Km values (and those shown

in Table 7) obtained in oocytes should be interpreted with transporters subtypes (454); this is consistent with thefour mammalian brain EAAT transporter isoforms iso-caution since they depend on the expression system used:

human EAAT2 has an apparent Km for L-glutamate of 18 lated.The zwitterionic amino acid transporters of this su-mM in oocytes (19), whereas for rat EAAT2 (i.e., GLT1)

expressed in HeLa cells, the Km is 10 mM, and 2 mM when perfamily share as general transport properties high affin-ity for zwitterionic amino acids and low affinity for L-it is reconstituted in proteolisosomes (424). This latter

value fits the affinity of L-glutamate when assayed in rat glutamate and sodium dependence, whereas the potas-sium dependence is controversial (20, 269, 488, 541, 627).brain membrane preparations or in rat brain purified and

reconstituted glutamate transporter (120, 255, 263, 425). A weak potassium dependence was described initially forASCT1 (alanine-induced current, Ref. 20) and ASCT2 (ala-Endogenous L-glutamate diluting the radiolabeled tracer,

membrane lipid composition, posttranslational modifica- nine uptake, Ref. 568) upon expression in oocytes. To ourknowledge, potassium dependence for ATBo has not beentions, or other factors may affect the apparent affinity of

these transporters in different expression systems (19, examined. Later on, Zerangue and Kavanaugh (627) statedthe contrary, i.e., that alanine transport (both influx and424). Interestingly, the apparent Km values determined for

rat EAAT1 in oocytes when measured by radiolabeled efflux) was independent on the external potassium con-centration in oocytes expressing ASCT1 under voltageglutamate (Ç80 mM) are higher than those based on mea-

surement of the induced glutamate-dependent current (11 clamp. One explanation of these dissimilar results, ob-tained with electric or radiolabeled L-alanine uptake stud-mM) (289), favoring the impression that endogenous sub-

strate diluting the radiolabeled substrate increases the ies, within the same group could be that alanine transport


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via ASCT1 is electroneutral, but sodium and the substrate ride (568). This suggests an electroneutral amino acid ex-change mechanism like that of ASCT1, and a not veryevoked a chloride channel activity associated with ASCT1,

which is thermodynamically uncoupled from the amino conspicuous ligand-evoked channel mode of action, butto our knowledge, amino acid exchange via ASCT2 hasacid transport (627). It has been suggested that the sodium

dependence of ASCT1 is not the result of sodium cotrans- not been examined. On the other hand, the lack of electricactivity of ASCT2 (627) might be a consequence of chargeport, since transport of L-alanine via ASCT1 is electroneu-

tral and potassium independent, and the ligand-evoked flux compensation between the transport mode of actionof the carrier and a yet unknown chloride channel associ-chloride current shows a Hill coefficient of 1 for external

sodium (627). In contrast, the electroneutral uptake of ated activity? If so, what is the electric activity of ASCT2in a chloride-free system (i.e., extracellular chloride Å 0radiolabeled L-alanine via ASCT1 involves the electroneu-

tral exchange of extracellular and intracellular amino acid and chloride-depleted oocytes)?4) Finally, is the ligand-evoked chloride channel ac-substrates (627); influx and efflux of L-alanine gave a 1:1

flux stoichiometry with the substrate specificity of ASCT1. tivity associated with the glutamate transporters of thissuperfamily and to ASCT1 extensive to ASCT2 and ATBo?Interestingly, external sodium is needed for this exchange.

Very recently, Ganapathy and Leibach (162) reported ex- The ligand-evoked current associated with rabbit ATBo

reverses at 030 to 040 mV (270). Is this a reflection ofchanger transport activity for ATBo, but a full paper isstill lacking on this issue. In addition, a substrate-induced an associated ligand-evoked channel activity? If so, its ion

selectivity might be different from the channel activitycurrent that reverses at 030 to 040 mV has been associ-ated with rabbit ATBo (270). These results offer a new associated with the other members of this superfamily

that tends to reverse current at the equilibrium potentialview of the transport mechanisms of these zwitterionicamino acid transporters: 1) electroneutral exchange of of chloride in oocytes (i.e., 023 mV).

Transporters ASCT1 and ASCT2 upon expression inzwitterionic amino acid which is dependent on extracellu-lar sodium; 2) ligand-evoked chloride currents, which are HeLa cells or oocytes have transport properties similar to

system ASC (185) (see Table 7), i.e., high-affinity (micro-thermodynamically uncoupled from the amino acid trans-port, as for the EAAT transporters of this superfamily; molar range) and sodium-dependent transport for neutral

amino acids (including small ones like alanine, serine, andand 3) lack of interaction with potassium. Interestingly,these zwitterionic amino acid transporters do not con- cysteine) and interaction with anionic amino acids at pH

õ7.4 (i.e., the protonated amino acid species is probablyserve the glutamate residue identified as crucial for potas-sium countertransport of the EAAT transporters of this the substrate), and insensitive to inhibition by dibasic

amino acid and the analog MeAIB (20, 488, 541, 568).superfamily (see sect. IIC7). Additional studies are neededto clarify these transport mechanisms. This suggested that these two carriers represent isoform

variants of system ASC. Indeed, system ASC showed sig-1) Does ASCT1 operate as an obligatory exchangerfor a wide range of amino acid and sodium gradients nificant variability in substrate specificity; e.g., threonine

is a better substrate than cysteine in rat liver, but thethrough the plasma membrane? Unfortunately, in the Zer-angue and Kavanaugh’s study of the amino acid exchanger converse is true in the hepatoma cell line HTC (165, 196,

275, 569). Northern analysis showed a very low expressionactivity of ASCT1 (627), influx and efflux rates were onlycompared at a fixed concentration of external L-alanine of ASCT1 and no expression of ASCT2 in liver, a tissue

with high system ASC activity. This suggests that neitherand sodium; it is therefore possible that this exchangereflects ASCT1 transport near equilibrium and not a real ASCT1 nor ASCT2 represents the liver system ASC trans-

porter. Work by Kilberg’s group aiming to identify a liverobligatory exchanger mechanism. A similar transportmechanism, but concentrative, was described for system homolog of ASCT1–2 failed to isolate a new transporter

cDNA (M. S. Kilberg, personal communication). SystemASC in fibroblasts (76).2) What is the role of sodium in the ASCT1 exchanger ASC is concentrative, voltage dependent, and electroneu-

tral (185). It is necessary to clarify whether ASCT1 andmode of action proposed by Zerangue and Kavanaugh(627)? Is the sodium ion really translocated through the ASCT2 mediate a concentrative transport (see above) of

their substrates before ascribing ASCT1 and ASCT2 trans-membrane, or is its action like that of an allosteric modu-lator? Uptake measurements of 22Na are needed to clarify porters to variants of system ASC.

The transport characteristics of ATBo upon expres-this issue. Then, if sodium is translocated through ASCT1,what mechanism explains the lack of concentrative trans- sion in HeLa cells and oocytes, as well as the epithelial

distribution of ATBo transcripts (269), suggested that thisport activity of ASCT1 located along the large sodiumelectrochemical gradient of the plasma membrane? transporter corresponds to the epithelial system Bo, the

major apical sodium-dependent transport for zwitterionic3) Is the exchange mechanism of transport of ASCT1and ATBo extensive to ASCT2? It has been reported that amino acids with broad specificity (excluding methylated

amino acids and dibasic and anionic amino acids) (329,mouse ASCT2 shows no electric activity evoked by aminoacid substrates and sodium even in the presence of chlo- 337, 515). The new view that ATBo and ASCT2 might be


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species counterparts of the same transporter with an elec- turally related substrate that is transported as a neutralzwitterion (626), resulted in a clearly lower intracellulartroneutral amino acid exchange mechanism of transport

(162, 270; V. Ganapathy, personnal communication) com- acidification than transport of an equivalent amount ofglutamate. Interestingly, this is at odds with evidence thatpromises the initial expected role of ATBo in the active

epithelial uptake of neutral amino acids. countertransport of anions (such as OH0 or HCO03 ) is

responsible for the pH-changing activity of glutamatetransport in salamander retinal glia cells (63).3. Stoichiometry

4) Countertransport of potassium is coupled with glu-tamate transport because, in chloride-free medium andThe stoichiometry of glutamate or zwitterionic amino

acids and the corresponding coupled ions, as well as the chloride-depleted oocytes expressing human EAAT3, su-perfusion of glutamate and sodium produced an inwardparticipation of pH-changing ions in the transport cycle, var-

ies among the members of the present superfamily. In gen- current, and superfusion with potassium produced an out-ward current; both currents are inhibited by 5 mM KA, aeral, for many cell types, glutamate uptake is electrogenic

and driven by cotransport of sodium and countertransport competitive antagonist of EAAT isoforms (see sect. IIC2).Changes in the reversal potential caused by altering gluta-of potassium with a first-order dependence on external L-

glutamate and internal potassium and a sigmoidal depen- mate and potassium membrane gradients are consistentwith the countertransport of potassium and glutamate.dence on external sodium, which suggests a stoichiometry

of 1 glutamate:3 sodium:1 potassium (30, 263). In addition, 5) By applying the changes in the glutamate transportreversal potential due to varying glutamate and ion gradi-movement of pH-changing ions (cotransport of H/ or count-

ertransport of OH0) occurs during transport (63, 141, 392). ents (i.e., for Na/, H/, or K/) to a zero-flux equation relat-ing the membrane potential to the transmembrane ionIn this instance, the stoichiometry of 1 glutamate:2 sodium:1

potassium:1 OH0 would still be electrogenic. As discussed gradients, the coupling coefficient for each substrate wassimilar to 1 glutamate:1 hydrogen:3 sodium:1 potassium.by Kanner (252), a stoichiometry of 1 glutamate:¢2 sodium

is favored by direct experimental evidence obtained by ki- 6) Consistent with the previous data, the Hill coeffi-cient for external sodium concentration at a fixed externalnetic and thermodynamic methods (141, 510).

The stoichiometry of the cotransported ligands of glutamate concentration (10 mM) was between two andthree, whereas this was not different from one when theEAAT transporters has been examined for rat EAAT1

(289) and human and rabbit EAAT3 (246, 628) expressed external concentrations of glutamate, hydrogen, and po-tassium were varied.in oocytes, and the models proposed are different for

each. Zerangue and Kavanaugh (628) offered evidence 7) This stoichiometry is consistent with a measuredinward positive charge transfer of two during uptake ofthat the neuronal human EAAT3 cotransports one gluta-

mate, one hydrogen, and one sodium and countertrans- tracer L-glutamate (100 mM) under voltage clamp.8) Finally, uptake of tritiated 1 mM L-glutamate inports one potassium, with a glutamate-to-charge ratio of

1:2 (2 positive charges accompany the movement of gluta- oocytes expressing human EAAT3 reaches a steady stateof õ10 nM external glutamate concentration, which withmate and in the same direction), and with this flux-cou-

pling model, a transmembrane gradient of glutamate an estimated intracellular concentration of anionic aminioacids in oocytes of 12 mM (626) represents a transmem-through the transporter of 106 is predicted. This is based

on the following. brane concentration gradient ú106; this value is compati-ble with cotransport of three sodium per glutamate, but1) Because of the uncoupled chloride channel activity

associated with the expression of human EAAT3 in oo- not with two sodium per glutamate.Surprisingly, Zerangue and Kavanaugh’s data (628)cytes (582), this study was conducted in a chloride-free

medium and chloride-depleted oocytes, or with oocytes on human EAAT3 are different from those previously ob-tained in rabbit EAAT3-expressing oocytes by Hediger andclamped at chloride equilibrium potential (025 mV) to

avoid the contribution of this channel activity to the fluxes co-workers (246). Initial studies aiming to elucidate thestoichiometry of rabbit EAAT3 revealed a Hill coefficientcoupled to the transport of glutamate.

2) Flux of the pH-changing ion is thermodynamically for external sodium that is dependent on the externalconcentration of glutamate, suggesting that the affinity forcoupled to transport, and it is not the consequence of

permeation through the uncoupled chloride channel activ- the sodium sites depends on the glutamate concentration;interestingly, the Hill coefficient was 2 at 200 mM gluta-ity of the transporter (137, 144, 295, 582), because in volt-

age-clamp conditions uptake of glutamate resulted in in- mate and ú2 at 10 mM glutamate (246). This covers theglutamate concentration range studied by Zerangue andtracellular acidification of the oocyte even with an in-

wardly directed electrochemical gradient for OH0. Kavanaugh (628). Parallel measurements of 22Na and[14C]glutamate flux, intracellular pH changes, and sub-3) Most likely, hydrogen is cotransported with gluta-

mate as a carboxylate ion pair, instead of the counter- strate-evoked currents gave a stoichiometry of cotrans-port of one glutamate for two sodium ions, countertrans-transport of OH0, because transport of L-cysteine, a struc-


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port of one potassium ion, and either cotransport of one membrane vesicles from rat brain demonstrated that in-flux or efflux of glutamate is coupled to efflux or influxhydrogen or countertransport of one hydroxide (246). Un-

fortunately, these studies were conducted without clamp- of potassium, respectively (261); the same situation hasbeen demonstrated with salamander retinal glia cells (23,ing the oocyte membrane potential. In any case, it will be

interesting to determine the accumulation capacity of 529). In addition, in the absence of potassium, the gluta-mate transporter catalyzes exchange of its anionic aminoboth EAAT transporters at different glutamate concentra-

tions (this has not been reported for the rabbit counter- acid substrates (255). This suggested that the glutamatetranslocation step is distinct from that of potassium. Thepart), as well as 22Na uptake measurements and determi-

nation of ion coupling coefficients at high glutamate con- glutamate transport cycle occurs in two parts: 1) translo-cation of sodium and glutamate, and 2) reorientation ofcentration (e.g., 200 mM and up) for the human

counterpart. the binding sites upon binding and translocation of potas-sium (252). When a comparison was made of the ion de-Consistent with Kavanaugh’s stoichiometry for hu-

man EAAT3, the glial rat EAAT1 expressed in oocytes pendence of net flux with that of exchange, the bindingorder of the substrates during influx was shown as followsshowed a Hill coefficient for the kinetics of external so-

dium and glutamate of approximately three and approxi- (255, 425): 1) an ordered binding of the coupled two orthree sodium ions before the binding of one glutamatemately one, respectively (289). In contrast to human

EAAT3, the current associated with glutamate transport ion to the extracellular face, 2) translocation of the com-plex, 3) release of glutamate and the sodium ions (it isvia rat EAAT1 was not changed by reducing external pH

from 7.4 to 6.0, suggesting that glutamate is transported not clear whether the release or binding at cytosolic faceis also ordered or random), 4) binding of potassium onas an anion (289). These results allowed Stoffel’s group

(289) to propose a stoichiometry for rat EAAT1 of 1 gluta- the inside, and 5) translocation and release of potassiumto start a new cycle. If necessary, the glutamate transport-mate, 3 sodium/1 potassium. This is at odds with the in-

creased glutamate transport current obtained by decreas- coupled pH-changing ions could be translocated concomi-tantly with either glutamate (i.e., hydrogen) or potassiuming the external pH in glial cells and in oocytes expressing

the neuronal human EAAT3 (48, 628). In contrast, it is in (i.e., hydroxide) steps (252). Additional evidence for thetranslocation of sodium and potassium ions in differentagreement with data in fibroblasts and salamander glial

cells (165, 482). This result might reflect the participation steps comes from the recent demonstration that ratEAAT2 Glu404Asp mutant is able to mediate exchangeof different pH-changing ions for glutamate transporters

of glial origin. In fact, studies of electrogenic uptake of of D-aspartate and sodium but not countertransport ofpotassium (266). The kinetics of human EAAT2 have beenglutamate into glial cells gave Hill coefficients between 2

and 3 (31, 482). In this sense, it will be very interesting examined upon expression in oocytes by analysis of non-linear capacitance (i.e., pre-steady-state or transient cur-to know if the stoichiometry and the pH-changing ions

for the glial EAAT2 fit the data reported by Attwell and rents) (583), after the pioneer work of Wright and co-workers (411, 412), who aimed to analyze the conforma-co-workers (63) in salamander retinal glia cells. To our

knowledge, these studies have not been reported either tional changes and kinetic properties of the sodium/glu-cose cotransporter. Human EAAT2 transient currents arefor EAAT2, EAAT4, or the retinal EAAT5.

Initial data on the stoichiometry and ion dependence compatible with the free carrier being in voltage-depen-dent equilibrium with the sodium-transporter complexof the zwitterionic amino acid transport via ASCT1 and

ASCT2 indicate variability of ion coupling among these and to a charge movement due to the binding of sodiumto a site of the carrier within the membrane electric fieldtransporters. Recent strong data supporting the idea that

human ASCT1 is a sodium-dependent zwitterionic amino (583). Again, EAAT isoforms show different mechanismsof transport, since these currents are not detectable withacid transporter exchanger (with a 1:1 stoichiometry of

exchange) might help to clarify this issue (see above; Ref. rabbit or human EAAT3 expressed in oocytes (see below;Refs. 246, 249). Analysis of the charge movement esti-627). Because of the lack of precise information on the

potassium dependence of ASCT2 and the mechanism of mated a glutamate turnover of 4–27 s01 over the voltagerange 0 to 0140 mV (583). Interestingly, these values aretransport of ASCT2 and ATBo, the suggested substrate

stoichiometry (568) is at present speculative. similar to those estimated with reconstituted glutamatetransporters from rat brain, which were the basis for thecloning of rat EAAT2 (120). In contrast, these values are4. Mechanism of transportsmaller than those obtained with salamander retinal gliacells (ú103 s01) (482). Again, this most probably reflectsThe mechanism of substrate translocation has been

studied in membrane preparations of neural origin by Kan- that these cells express EAAT transporters other thanEAAT2 (e.g., the EAAT5 isoform).ner’s group (for review, see Ref. 252), for the expressed

rabbit and human EAAT3, human EAAT2, and human Hediger and co-workers (246, 249) examined themechanism of transport for the rabbit and human neu-ASCT1 (246, 249, 583, 627). Studies with synaptic plasma


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ronal EAAT3 glutamate transporters. Expression in oo- ior of EAAT3 using cysteine as a substrate, the strategyused by Zerangue and Kavanaugh (628) to identify hydro-cytes of these two EAAT3 counterparts by two indepen-

dent labs demonstrated that the reverse mode of action gen as the pH-changing ion in EAAT3, and the behavior ofEAAT3 (e.g., pre-steady-state currents, potassium-specific(glutamate efflux; potassium influx) is a true reversal of

the overall forward reaction (246, 628); thus both reac- current-voltage relationship, and potassium Hill equation)in the reverse mode of action (i.e., absence of glutamatetions have the same stoichiometry, basic mechanism of

transport, and rate-limiting step (246), in agreement with and presence of potassium in the external medium).the model proposed by Kanner (252). Pre-steady-state cur-rents, substrate-specific current-voltage relationship, and 5. Ligand-gated chloride channel activity associatedsodium Hill equation-extracellular glutamate concentra- with the transporterstion relationship studies with human and rabbit EAAT3expressed in oocytes (246, 249) allowed Hediger and co- As stated in the introduction to this section, for the

human EAAT1–5 isoforms and the human zwitterionicworkers to propose that 1) at low extracellular sodiumconcentration (less than Km) binding of sodium to the amino acid transporter ASCT1, evidence for a mode of

action as a sodium/amino acid-gated chloride channel hasextracellular face of the transporter becomes rate lim-iting. 2) At high extracellular sodium concentration (more been offered by Amara’s and Kavanaugh’s groups (18, 144,

582, 583, 627; for review, see Ref. 507). In a recent review,than Km), the rate-limiting translocation step depends onthe extracellular L-glutamate concentration. At low con- Wright et al. (609) argued that this, in addition to a mode

of action as water channel of the sodium-glucose SGLT1centration (less than or equal to mean affinity constant;the apparent L-glutamate affinity depends on the mem- cotransporter and water cotransport of SGLT1 and GAT1,

and sodium leak by glutamate transporters and SGLT1,brane voltage, Refs. 249, 246), L-glutamate binding to theextracellular face becomes rate limiting, whereas at high GAT1, and NET1, is indicative of the multifunctional (i.e.,

cotransporters, uniporters, channels, and water transport-concentration of extracellular glutamate (more than meanaffinity constant), the charge translocation step becomes ers) behavior of these cotransporters.

Let us examine the evidence for the sodium/aminorate limiting (i.e., translocation of the fully loaded carrier,the carrier plus L-glutamate plus 2 sodium ions). These acid-gated chloride channel activity of human glutamate

EAAT1–5, rat EAAT2, and human ASCT1 transporters (18,results are consistent with previous studies on the high-affinity L-glutamate transport in renal brush-border mem- 144, 266, 582, 627).

1) The initial observation was that the cotrans-brane vesicles (208). In addition, cooperativity of sodiumand glutamate binding for rabbit and human EAAT3 porter-coupled currents were larger than expected for

the cotransporter density and turnover number, i.e., thestrongly suggests an ordered mechanism of binding: firstone sodium binds with low affinity, second glutamate current exceeds the charge movement due to the amino

acid transport. In chloride-depleted conditions or at thebinds, and third the second sodium binds with high affin-ity. The transport cycle then progresses as in the model chloride equilibrium potential (023 mV in oocytes), the

current due to the transport mode of action could beof Kanner (252). The authors hypothesize that the sodiumleak (i.e., transport of sodium in the absence of glutamate) estimated and would represent, at a membrane potential

of 060 to 080 mV, only 5% of the total current elicited(see Fig. 6) through EAAT3 is a consequence of the trans-location of the carrier with two sodium ions bound (246). by glutamate and sodium in oocytes expressing EAAT4

(144) and varies from 50 to 73% in oocytes expressingAll this suggests that the mechanism of the glutamatetransport cycle is different for the neuronal and renal human EAAT1–3 (582). In chloride-depleted conditions,

glutamate did not induce measurable current in oocytesEAAT3 transporter (246, 249) and the glial EAAT2 trans-porter (252). In one aspect, the model proposed by Hedig- expressing human EAAT5 (18). Thus it appears that cur-

rents elicited by glutamate and sodium in EAAT5 areer’s group for EAAT3 is in conflict with the suggestionthat this carrier transports glutamate as a carboxylate ion due primarily to the chloride conductance. For human

ASCT1, with an electroneutral mode of transport,pair together with one hydrogen ion, which acts as thepH-changing ion (628). Human and rabbit EAAT3 lack Ç100% of the ligand-evoked current is because of chlo-

ride conductance (627).significant current relaxation in response to voltage jumps(246, 249). This suggests that the empty EAAT carrier 2) In steady-state flux condition measurements, the

reversal potential (Erev) evoked by the amino acid andis electroneutral (i.e., any conformational change of thecarrier during the transport process does not involve sodium due to the expression of the transporters fits with

the proportion of chloride currents and the electrogenicitymovement of charge residues within the membrane elec-tric field) and that the translocation complex (i.e., carrier due to the amino acid transport activity (144, 582, 627,

18) (see Table 7): Erev values for EAAT4, EAAT5, andplus glutamate plus 2 Na/) has a positive charge and therelocation complex (i.e., carrier plus K/ plus OH0) is elec- ASCT1 are very close to the chloride equilibrium potential

in oocytes, whereas for EAAT1–3, Erev is within a positivetroneutral. It will be interesting to know the kinetic behav-


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FIG. 6A. 10 TM transmembrane topology model for rat EAAT2 transporter. EL and IL loops are numbered, accordingto model proposed by Slotboom et al. (498). Amino acid residues conserved in all known transporters of this superfamilyare indicated in gray circles, and residues that are specific for EAAT transporters are indicated in white over a blackcircle. Nine residues (in IL1, IL2, TM V, TM VI, IL3, TM VII, and TM X) and two glycosylated (Y) Asn residues in EL2are marked by a square, indicating homologous residues that, after analysis by site-directed mutagenesis studies, havebeen shown to be critical for transport activity (see text for details). Shaded area delimits protein segment involved inkainate binding for human EAAT2 (571). To our knowledge, gene structure of rat EAAT2 has not been reported. Codingregion of human EAAT2 gene is composed of 10 exons (360; not yet accessible in data bases). Human EAAT1 gene iscomposed of 10 exons (accession numbers in NCBI: Z31713, Z31703-Z31710; W. Stoffel, M. Dueker, R. Mueller, and K.Hofmann).

range of membrane potential. The lowest chloride con- kinetic analysis of the amino acid flux via EAAT5 has notbeen reported.ductance contribution is found with EAAT2, for which

current reverses only at /60 mV. 5) For all these transporters, the sodium/amino acid-evoked chloride conductance exhibits the same chao-3) The chloride current is thermodynamically uncou-

pled from the amino acid flux via these transporters (18, tropic selectivity sequence: SCN0 ú NO03 ú I0 ú Cl0 (144,

582, 627); for human EAAT5, only the partial sequence144, 582, 627). Thus, for all these transporters, radiola-beled amino acid flux is independent of the chloride cur- NO0

3 ú Cl0 has been reported (18).rent direction and the magnitude of the anion flux when What is the nature of this sodium/amino acid-evokedsubstituting chloride, and for EAAT4 and ASCT1, it is chloride current? All the data could be interpreted as anlargely or totally independent of the membrane potential. intrinsic chloride channel activity of these transporters

gated by sodium and the amino acid or, alternatively, as4) The chloride conductance is evoked by sodiumand the amino acid substrate (18, 144, 582, 627), and with the induction, via direct or indirect interaction, of a silent

chloride channel of the oocyte. The ion selectivity of theidentical apparent affinities as in the transport mode ofaction of the corresponding transporters (144, 582, 627); chloride conductance associated with these transporters


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FIG. 6B. 6 TM a-helix plus 4 TM b-sheet transmembrane topology model for human ASCT1 transporter. Aminoacid sequence presented corresponds to that of human ASCT1. EL and IL loops are numbered according to modelproposed by Wahle and Stoffel (585) for rat EAAT1. Amino acid residues conserved in all known transporters of thissuperfamily are indicated in gray circles, and residues that are specific for zwitterionic transporters are indicated inwhite over a black circle. Y, glycosylated residues. Shaded area delimits protein segment homologous to that involvedin kainate binding for human EAAT2 (571). Exon boundaries within putative protein sequence are indicated, and exonsare numbered (214).

is different from that of the endogenous calcium-depen- that an intermediate complex of the amino acid transportcycle activates an endogenous silent chloride channel. Indent chloride channel activity of the oocytes, and it is

not inhibited by typical oocyte chloride channel blockers this hypothesis, the identical ion selectivity of this chlo-ride channel activity for many of the transporters of this(144, 582). These data indicate that the chloride channel

conductance associated with these transporters is not due family would reflect the activity of a single type of chan-nel, and the different magnitude of the chloride channelto activation of known endogenous chloride channels of

the oocytes. Interestingly, for EAAT4, the Vmax of transport activity would be due to the activation of a differentialnumber of chloride channels by these transporters. In thisfor radiolabeled L-glutamate is higher (õ2-fold) than that

for L-aspartate, whereas the maximum current of the asso- instance, the differential chloride current due to L-aspar-tate and L-glutamate in EAAT4-expressing oocytes mightciated chloride channel for L-aspartate is higher (3-fold)

than that for L-glutamate (144). This has been interpreted indicate that activation of the chloride channel occursthrough interaction with a particular intermediate com-as showing that gating of the channel occurs after binding

of the sodium ions and the amino acid, and the different plex of the transport cycle that would become more repre-sented in the steady-state obtained with L-aspartate thansize of L-glutamate and L-aspartate allows a larger chloride

flux in the latter case (144, 582). the one obtained with L-glutamate. This transport com-plex might be the translocating complex (carrier plus so-In our view, however, there is still room for activation

of an endogenous silent chloride channel. It is conceivable dium/amino acid). Notice that the associated chloride


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channel activity of the potassium independent amino acid ionic dependence) (175). 2) Presynaptically, the activa-tion of a chloride conductance concomitant with gluta-exchange via ASCT1 (627) and via the Glu404Asp EAAT2

mutant (266) points to this intermediate. As discussed mate transport would provide a potential mechanism tooffset the depolarizing action of transmitter reuptake andabove for the neurotransmitter transporter superfamily,

only a small fraction of the expressed GAT-1 and NET reduce cell excitability. Thus, in salamander cone photo-receptors, a glutamate-evoked chloride conductance, withcotransporters behaves as ion channels in the presence

of the ligands (GABA or norepinephrine and Na/) (82, properties similar to the glutamate transporters, respondsas an inhibitory signal (hyperpolarization) to the release160). This could be interpreted as an extremely low open

probability of the channel (reviewed in Ref. 609) or as of glutamate from the same cell (421). The physiologicalrelevance of the thermodynamically uncoupled chloridea low fraction of the transporters interacting with the

endogenous silent channels. In our view, reconstitution conductance of the glutamate transporters in several celltypes (retinal and pituitary cells) has recently been re-of the sodium/amino acid-gated chloride channel activity

in proteoliposomes containing expressed and purified viewed by Sonders and Amara (507).EAAT or ASCT1 transporters may be the final demonstra-tion of the intrinsic channel activity of these transporters. 6. Protein structureMore simply, it will be interesting to know the relationshipbetween amino acid transport rate and the chloride con- The main common structural features (Figs. 5 and

6) among the mammalian members of this family are asductance at different levels of transporter expression.For two members of the present superfamily, ASCT2 follows: 1) the absence of a cleavable signal sequence,

suggesting a cytosolic localization of the NH2 terminus;and ATBo, no chloride channel activity has been de-scribed, but to our knowledge, this has not been properly 2) the absence of an SOB motif, identified in a variety

of sodium/solute cotransporters; 3) the presence of thetested (269, 568). Very recently, the amino acid-evokedcurrent associated with ATBo has been reported to reverse sequence motif AA(I,V,L)FIAQ, probably located in a

membrane-spanning domain, which is conserved through-at 030 to 040 mV (270). At present, there is no clearexplanation for this Erev value in terms of an associated out the evolutionary diversity of glutamate transporters

from prokaryotes to mammals, and also in the zwitterionicchloride conductance. Data from two different labs (Hed-iger’s and Stoffel’s groups) are in apparent contradiction amino acid transporters of this family; 4) a higher level

of conservation in the COOH-terminal half of the proteinswith the chloride channel activity associated with EAAT1and EAAT3. L-Glutamate-induced current due to rat which exceeds the level of conservation in the NH2-termi-

nal half by a factor of at least three; 5) the presence of sixEAAT1 expression in oocytes does not reverse up to /80mV (289), and that due to rabbit and human EAAT3 ap- highly conserved putative membrane-spanning domains in

the NH2-terminal half of the proteins; 6) the presence ofproaches asymptotically zero at /50 mV (246, 249). Isthis a consequence of a differential behavior of different two cannonical sites for N-linked glycosylation on a pre-

sumably extracellular hydrophilic loop EL2 between TMspecies counterparts (Erev for human EAAT1 is /9 mV,see Table 7; Ref. 582), or does it reflect differential experi- domains III and IV; and 7) a similar appearance of

EAAT1–3 glutamate transporters (there is no availablemental protocols? In the latter sense, it is worth men-tioning that Hediger’s group (246) did not show currents data for EAAT4 or the zwiterionic transporters) as broad

electrophoretic bands of similar size (65–75 kDa) due toat depolarization potentials over /50 mV, and Amara andKavanaugh and co-workers (582) measured an Erev of /38 variable glycosylation (121, 298, 305, 461, 481).

Ever since the initial description of the first threemV for human EAAT3.Although the mechanism of the uncoupled chloride members of this superfamily (EAAT1–3), the topology

of these transporters in the plasma membrane has beenconductance during the transport cycle remains un-known, there is compelling evidence for a chloride con- controversial. Based on topology algorithms, Stoffel’s lab

(214, 518) for EAAT1 and ASCT1, Kanner’s group (424)ductance in parallel with sodium/glutamate cotransportin photoreceptors and bipolar cells, where the ligands for EAAT2, and Hediger’s lab (245) for EAAT3 agree on

the presence of six classical a-helix TM domains in theincrease the rate of opening of the chloride channel (137,138, 175, 176, 295, 471, 531). The retinal EAAT5 trans- first NH2-terminal part of these proteins (see Fig. 5). Con-

troversy appears in the COOH-terminal part, and this isporter, with a putative synaptic localization (see sect.IIC1), may be responsible for this chloride conductance. an important issue since homology in this part of these

proteins is very high (see Fig. 5) and several amino acidPre- and postsynaptic glutamate-gated chloride conduc-tances may have physiological roles in vertebrate retina. residues critical for transport activity have been described

within this region (see sect. IIC7). There is a long hy-1) The light response mediated by cones in depolarizingbipolar cells in the perch retina is due to the closing of a drophobic stretch of amino acid residues with no clear

tendency to show a-helix structures toward this end inpostsynaptic chloride conductance that has properties ofthe glutamate transporter (i.e., similar pharmacology and any of the members of this superfamily (dashed line in


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Fig. 5). Kanner’s group (424) suggested the presence of not attempt to demonstrate that these chimeras are ex-pressed in the plasma membrane and not as inclusiontwo additional classical TM domains within this protein

region for EAAT2, whereas Kanai and Hediger (245) in- bodies. In addition, the model proposed is very rigid andneeds three very small loops (EL4, IL4, and EL5) whichcluded four additional classical TM domains. In contrast,

Stoffel and co-workers (214, 518) suggested six classical is difficult to apply to mammalian members of this super-family because of the presence of charged residues at theTM domains and four hydrophobic b-sheets crossing the

plasma membrane. In all cases, these models positioned extremes of TM domains VII, IX, and X (see Fig. 6A).Stoffel’s model is based on very clear data for the firstthe COOH terminus inside the cell. Very recently, two

studies offered experimental evidence on the topology of NH2-terminal part of the protein (TM domains I-VI); allthe chimeras constructed showed glycosylation of the re-EAAT1 (585) and the glutamate transporter GltT from

Bacillus stearothermophilus, a prokaryote-related mem- porter protein domains (they used an endogenous N-gly-cosylated domain of EAAT1 that corresponds to a largeber of this superfamily (498). After these studies, the con-

troversy remains. Stoffel and co-workers (214, 518) ap- portion of the EL2 loop) when connected to loops EL1,EL2, and EL3. Conversely, the reporter protein domain isplied ‘‘reporter glycosylation scanning’’ (i.e., chimeras

containing EAAT1 domains and an N-glycosylated re- not glycosylated when located in loops IL1, IL2, and IL3(see Fig. 6, A and B). This part of the model is confirmedporter peptide), expressing chimeras in oocytes, to sup-

port a model for EAAT1 (GLAST-1) with 10 TM domains by the following evidence: 1) the NH2 terminus is intracel-lular since EAAT1-specific antibody immunofluorescence(see Fig. 6B). This model has NH2 and COOH termini

intracellular, six NH2-terminal hydrophobic TM a-helices, signal is only obtained with permeabilized cells (298, 585).2) For EAAT1, Stoffel’s group showed by peptide sequenc-and four COOH-terminal short hybdrophobic domains

spanning the membrane bilayer as b-sheets. The six NH2- ing, endoglycosidase F treatment, and site-directed muta-genesis that Asn-206 and Asn-216 residues are the onlyterminal hydrophobic TM a-helices correspond to those

suggested for all these transporters. Site-directed antibod- ones in the whole protein sequence that are N-glycosyl-ated (116, 481); these residues are located in the extracel-ies used in immunofluorescence studies with permeabil-

ized cells confirmed the intracellular location of the NH2 lular loop EL2. 3) The Ser-113 residue of the glutamatetransporter EAAT2 is phosphorylated in vivo by proteinterminus of EAAT1 (585) and the COOH terminus of rat

EAAT1 and EAAT2 (298), suggesting an even number of kinase C (83); this agrees with the intracellular locationof the IL1 loop (see Fig. 6A).TM domains. Slotboom et al. (498) used alkaline phospha-

tase (PhoA) gene fusion technique (i.e., scanning chimeras In contrast, Stoffel’s model of the topology for theCOOH-terminal part of EAAT1 (585) is based on data thatcontaining GltT domains and alkaline phosphatase) to

study the controversial COOH-terminal part of the pro- appear to be inconsistent. The ‘‘reporter glycosylationscanning’’ data obtained with chimeras constructed withkaryotic GltT transporter. Extrapolation of their results

to the eukaryotic members of the superfamily is war- residues located in the proposed intracellular loops IL3and IL4 and the COOH-terminal domain are clearly consis-ranted by the fact that all these transporters showed a

very similar hydropathy profile in the COOH-terminal half tent with the model, but those with residues located inthe proposed extracellular loops EL4 and EL5 are contro-of the protein (i.e., fragment comprised between amino

acid residues 400 and 550 of the multialignment of versial. 1) The latter chimeras produce only Ç50% of theprotein with the reporter domain glycosylated. In addi-EEAT1–4 isoforms and GltT from E. coli, Bacillus sub-

tillis, and B. stearothermophilus) (498). The GltT topol- tion, the fusion protein of the reporter glycosylation do-main at a residue located in the proposed EL4 loop is notogy model proposed the presence of four additional TM

a-helices in the COOH-terminal half of the protein (see glycosylated at all in the reporter domain when expressedin oocytes. To explain these results, the authors need toFig. 6A) (498).

Both strategies have been used to study the mem- invoke reorientation or steric hindrance for the transloca-tion of the COOH-terminal reporter domain to the lumenbrane topology of several proteins and are considered a

good technical standard. In our view, however, both stud- of the endoplasmic reticulum because of the moderatesize and hydrophobicity of TM domains VII and IX actingies (498, 585) lack clarity, contain inconsistent data, and

in addition used an objective experimental strategy to as anchoring sequences (see Fig. 6B). 2) A new N-glycosyl-ation site produced by site-directed mutagenesis in thefavor previous subjective topology models. Konings’

group (498) based their model on the expression of the center of the proposed extracellular loop EL5 is not glyco-sylated when expressed either in oocytes or in a transla-PhoA activity toward the periplasmic space if the particu-

lar chimera positioned PhoA extracellularly. The expres- tion system in vitro (585). 3) Three GltT-PhoA fusion pro-teins in amino acid residues within GltT protein regionssion of low PhoA activity is interpreted as an intracellular

location of the Phoa domain in the chimeras. In all cases, that are homologous to the extracellular loops EL4 andEL5, proposed by Stoffel’s group, gave rise to a very lowthey demonstrate that low PhoA is not caused by a low

expression level of the particular chimera, but they do periplasmic PhoA activity (498).


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Finally, it is interesting to notice that the two groups superfamily, using chimeric proteins (between the humanhomologs of EAAT1 and EAAT2; Ref. 571), site-directedgave differing interpretations to results that are consistent

with each other. For instance, the higly conserved motif mutagenesis (for rat EAAT1 and EAAT2 transporters;Refs. 83, 115, 116, 426, 630), on the conformationalAA(I,V,L)FIAQ is placed in Stoffel’s b-sheet TM domain

IX and in Konings’ a-helix TM domain VII. This is based changes associated with the transport step (for ratEAAT2; Refs. 179, 266, 583), or on the homomultimeriza-on 1) a nonglycosylated reporter domain and a low peri-

plasmic PhoA activity when the reporter domain is fused tion of these transporters (for rat EAAT1–3; Ref. 199).Part of these studies has been recently reviewed (254).to EAAT1 Glu-406 residue (498) or to its homologous resi-

due in GltT (585), 2) a low periplasmic PhoA activity when In an elegant study, Kavanaugh, Amara, and co-work-ers (571) prepared a human EAAT1–2-1 chimera, in whichthe fusion involves the GltT residue corresponding to

EAAT1 Ile-413, and 3) a partial glycosylated reporter do- 76 amino acid residues of EAAT2, comprising most of thehighly conserved long hydrophobic stretch (see Figs. 5main and a high periplasmic PhoA activity when the re-

porter domain is fused to EAAT1 Gln-425 residue (498) and 6A) were exchanged within the EAAT1 sequence. ThisEAAT2 protein segment, in which only 18 amino acid resi-or to the GltT residues that are homologous to the EAAT1

421 and 426 residues (585). This is used by Stoffel’s group dues are different in the two isoforms, corresponds topart of the IL3 loop, TM domain VII, EL4 loop, and mostto propose a b-sheet (residues 407–416 of EAAT1) and

by Konings’ group to propose an a-helix (corresponding of TM domain VIII in the 10 a-helix TM domain model(498) (see Fig. 6A). This segment in the EAAT1–2-1 chi-to residues 410–427 in the EAAT1 sequence) spanning

the plasma membrane, respectively. Stoffel’s group argues mera, when expressed in oocytes, confers sensitivity toinhibition by the nontransported competitive analog KAthat in their studies, most probably, there is no room

for an a-helix between the EAAT1 residues 407 and 425. to both glutamate transport (Ki in the micromolar range,characteristic of EAAT2 isoform) and to the uncoupledKonings’ group argues that detailed studies with the lac-

tose permease LacY and the melibiose carrier MelB from glutamate-independent sodium leak current, characteris-tic of EAAT1 isoform. Kinetic analyses are compatibleE. coli have demonstrated that the NH2-terminal half of

an outgoing TM helix is sufficient to export the PhoA with inhibition of both processes by binding of KA to asingle site (571). Interestingly, other transport characteris-domain fused to a membrane protein, whereas the NH2-

terminal half of an ingoing TM helix is sufficient to prevent tics of EAAT1 isoform are unchanged in the EAAT1–2-1chimera, like the apparent affinity for the substrate analogthe export of the PhoA moiety to the periplasm (77, 428).

It is patently clear that the topology of these trans- SOS (see Table 7) and the Erev of the glutamate- and so-dium-induced current [Erev Å Ç10 mV for EAAT1–2-1porters stands in need of further research. The two mod-

els are quite different and could be tested with alternative (461); compare with the Erev for EAAT1 and EAAT2 inTable 7]. This suggests that the kinetic parameters forstrategies. Studies with limited proteolysis and peptide-

directed specific antibodies could be informative. Notice substrate translocation and the uncoupled chloride chan-nel activity are determined by the EAAT1-derived se-that the exposed loops in the COOH-terminal half of these

transporters in Stoffel’s model are very conspicuous, quences.Most of the amino acid residues critical for the trans-whereas in Konings’ model they are very limited (see Fig.

6, A and B). Alternatively, vectorial labeling of cystine port function of EAAT1 and EAAT2 transporters revealedby site-directed mutagenesis are within or near this highlyresidues reintroduced in the borders of the proposed TM

domains of a cystineless transporter may also help. The conserved COOH-terminal part of glutamate transporters,which confers sensitivity to KA. Conradt and Stoffel (115)establisment of the membrane topology of the COOH-

terminal half of these transporters is an important issue analyzed the effect of substitution of three positivelycharged residues (Arg-122, Arg-280, and Arg-479) and onebecause of the high level of homology in this region for

all the members of this superfamily, and because several polar residue (Tyr-405) in rat EAAT1 transporter, whichare conserved in the glutamate transporters and substi-studies have shown that residues within this region are

critical for substrate binding or translocation (see sect. tuted by apolar residues in the zwitterionic transporters(see Fig. 5). Mutations Arg122Ile and Arg280Val (and bothIID). Finally, to date, b-sheet TM domains have been pro-

posed for several eukaryotic and prokaryotic membrane together) reduce the apparent affinity for L-aspartate with-out affecting the kinetic parameters for L-glutamate trans-proteins like the acetylcholine receptor, the VDAC ion

channel, and the lac permease (6, 52, 438), but they have port, and mutants Tyr405Phe and Arg479Thr, within thehighly conserved COOH-terminal part of these transport-only been demonstrated by X-ray analysis of the bacterial

porins (118). ers, completely abolished the intrinsic EAAT1 transportactivity (see below) (115). Kanner and co-workers (426)

7. Structure-function relationship analyzed the role in transport of five negatively chargedresidues of rat EAAT2 located in hydrophobic surround-Our knowledge of the structure-function relationship

is based on studies with glutamate transporters of this ings and highly conserved within the glutamate trans-


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porter family (Asp-398, Glu404, Glu461, Asp462, and Asp- cytes (266, 583) are hardly affected by the mutant (266).These transient currents are thought to be a reflection470). Only three of these residues (Asp-398, Glu-404, and

Asp-470; indicated in Fig. 6A) are critical for intrinsic of either sodium binding or a subsequent conformationalchange of the transporter. In agreement with the EAAT1–transport activity, which could not be explained by pro-

tein expression level or defects in trafficking to the plasma 2-1 chimera studies discussed above (571), the Glu404Aspmutant, located within the KA-binding/sensitive determin-membrane. Interestingly, defective transport cannot be

attributed to the mere requirement of a negative charge ing domain, does not affect the uncoupled amino acidsubstrate/sodium-induced chloride channel activity of theat this residues (i.e., transport is also affected by substitu-

tion of the corresponding charged residue, either Glu or transporter (266), suggesting that this protein region doesnot influence this channel activity. It is remarkable thatAsp) (426).

The rat EAAT2 Glu404Asp (this residue is located in Glu-404 residue is in between two other conserved resi-dues in all glutamate transporters of the family, compris-the TM domain VII of the 10 a-helix TM domain model;

see Fig. 6A) mutant has been revealed as a powerful tool ing the sequence Tyr-Glu-Ala (see Fig. 5). Interestingly,the Glu404Asp homologous mutation in human EAAT3to address structure-function studies. This mutant con-

serves most of D/L-aspartate transport (Ç80%), but only also abolishes potassium-dependent efflux (266). In con-trast, the zwitterionic amino acid transporters of this fam-a small part of L-glutamate transport (õ20%) (426). The

defective Glu404Asp L-glutamate transport is not because ily have the conserved sequence Phe-Gln-Cys (see Fig. 5).Interestingly, mutation of this conserved Tyr residue toof defective binding (i.e., high-affinity L-glutamate inhibi-

tion of D/L-asparatate transport is conserved). This allows Phe (as in the zwitterionic transporters of this family) inrat EAAT1 (Tyr405Phe) abolished the intrinsic glutamatethe authors to propose that the Glu-404 (conserved in all

the glutamate transporter isoforms but absent from the transport activity (115), and in rat EAAT2 (Tyr403Phe)abolished interaction with potassium, and resulted in anzwitterionic transporters of this transporter family; see

Fig. 5) determines the amino acid substrate permeation increased sodium affinity (629a). Very recently, Zerangueand Kavanaugh (627) offered evidence that ASCT1 trans-pathway of the glutamate transporters. The Glu-404 resi-

due in EAAT2 together with residues Arg-122 and Arg-280 porter has an electroneutral exchange mode of transportfor the amino acid substrate and sodium through the mem-within the NH2-terminal part of EAAT1 (see above) are

those already identified, which are involved in substrate brane; this mechanism of transport has also been sug-gested for ATBo (162). It is therefore tempting to speculatespecificity discrimination (115, 426). Very recent data ob-

tained from collaboration between Kanner’s and Kava- that Glu-404 within these residues (located in the VII TMdomain in the 10 a-helix topology model, see Fig. 6A)naugh’s groups (266) showed that Glu-404 residue also

influences the potassium transport coupling (either bind- confers coupled cotransport of sodium and countertrans-port of potassium, whereas its lack determines an ex-ing or translocation), and the rat mutant Glu404Asp

EAAT2 catalyzes obligatory exchange of coupled amino change mode of transport coupled with sodium. Unfortu-nately, for human ASCT2 transporter, which also containsacid substrate and sodium through the plasma membrane.

1) The sodium/D-aspartate transport via Glu404Asp the conserved sequence Phe-Gln-Cys, the mechanism oftransport and potassium dependence has not been ad-EAAT2 is electroneutral in oocytes, 2) external potassium

does not reverse transport through Glu404Asp in oocytes, dressed in depth. In summary, the hydrophobic, topologi-cally controversial, and highly conserved domain locatedand 3) in the liposome, reconstituted mutant influx and

efflux of radiolabeled D-aspartate are dependent on trans- toward the COOH terminus of the glutamate transportersis involved in kainate binding and amino acid and ionsodium/amino acid substrate but not on trans-potassium.

In contrast, wild-type EAAT2 catalyzes trans-potassium- (potassium coupling) permeation pathways.Several residues within the NH2-terminal part of thesedependent influx and efflux of amino acid substrate in

the presence of sodium. This is a consequence of the transporters have been shown to be involved in theirtransport activity or expression (83, 116, 630), in additioncountertransport of potassium in the transport mecha-

nism of these glutamate transporters (255, 529; see sect. to the above-mentioned EAAT1 Arg-122 and Arg-280 resi-dues (115). Stoffel and co-workers (116) demonstratedIIC4). Because the Glu404Asp mutant is locked in an ex-

change mode of transport, either potassium binding or that the deglycosylated rat EAAT1 (N-glycosylation oc-curs in 2 canonical sites within the loop EL2; see Fig.permeation, or sodium binding is affected (i.e., a signifi-

cant increase in sodium binding will displace potassium 6A) is fully active, and none of its kinetic parameters isaffected. Kanner and co-workers (630) examined the ef-binding and force the transporter toward amino acid/so-

dium exchange) (266). The former possibility seems to be fect of substitution of the only two positively chargedresidues (Lys-298 and His-326 in EAAT2; see Figs. 5 andtrue because apparent sodium affinity is unchanged in the

Glu404Asp mutant. From all this, it is not surprising that 6A) conserved in all members of this transporter familyand located within putative a-helix TM domains (TM do-the sodium-dependent transient currents produced by

voltage jumps in human and rat EAAT2 expressing oo- mains V and VI in the 10 a-helix TM domain topology


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model; see Fig. 6A). Replacement of these residues by or potassium binding; these studies suggest that EAAT2transporter has at least two conformation states and thatsmall hydrophilic or positively charged amino acids pro-

duces in Lys-298 mutants a partial plasma membrane tar- the transition between them is associated with the trans-port step (179).geting defect and partial intrinsic transport defect of

EAAT2; His-326 mutants have an almost complete impair- Finally, EAAT1–3 glutamate transporters form homo-multimers (dimers and trimers), as revealed by chemicalment of their intrinsic transport activity without a traf-

ficking defect toward the plasma membrane (630). Zhang cross-linking in intact brain membranes and solubilizedtransporters, or after reconstitution in liposomes (199).et al. (630) suggested two possible roles for the conserved

His-326 residue. In analogy with structure-function studies The original EAAT2 purification studies by Kanner andco-workers (120, 121) revealed that the monomeric 73-of the proton-coupled lactose permease of E. coli, His-

326 could either form ion pairs with negatively charged kDa band of the transporter correlated with glutamatetransport activity. In addition, it is interesting that theresidues within TM domains that stabilize the transporter

(133, 283, 466) or participate in the mechanism of hydro- fully deglycosylated EAAT1, obtained either by deletionof the two glycosylation sites or after endoglycosidase Fgen transport (241, 406). The same group (426) examined

the first possibility by constructing double mutants with treatment, does not homodimerize in electrophoretic gels,and it is fully active (116). These data suggest that dimer-three conserved negatively charged residues that are criti-

cal for the transport activity of EAAT2 (Asp-398, Glu-404, ization of glutamate transporters does not affect theirtransport activity. In contrast, radiation inactivation stud-Asp-470; see above). None of the double mutants (i.e.,

His326Asn with Asp398Asn, Glu404Asn, or Asp470Asn) ies suggest that the minimal funtional unit correspondsto an oligomer of the rat EAAT2 transporter (199). It isregained activity, and therefore, there is no evidence for

these ionic pairs within EAAT2 transporter. therefore clear that further research is needed on thisissue.A very interesting line of research is the stimulation

of EAAT2 by protein kinase C. In loop IL1 of EAAT1–4glutamate transporters isoforms, there is a protein kinase 8. Physiological role of the glutamate superfamilyC canonical site (see Fig. 6A). Gimenez and co-workers transporters(84) showed that phorbol esters increased Vmax of sodium-glutamate cotransport in cultured glial cells. Later, these It is believed that the transporters in this superfamily

have a role both in the termination of transcription in theauthors in collaboration with Kanner’s group (83) demon-strated the following: 1) protein kinase C phosphorylates, synapsis and also in the supply of nutrients to brain and

peripheral tissues (205, 244, 247, 252, 269, 568, 627). Thein serine residues, pig brain purified glutamate trans-porter; 2) phorbol esters increase in parallel glutamate overall process of synaptic transmission, except for acetyl-

choline, is terminated by high-affinity sodium-dependenttransport activity (2-fold) and phosphorylation of immu-noprecipitated EAAT2 in C6 glial cells; and 3) rat EAAT2 transport of neurotransmitters (e.g., GABA, L-glutamate, gly-

cine, dopamine, serotonin, and norepinephrine; see reviewstransfected in HeLa cells is stimulated by phorbol esters,and mutation of Ser-113 to Asn abolished this stimulation in Refs. 205, 252, 253, 394). The concentration of L-gluta-

mate, the predominant excitatory neurotransmitter of thewithout affecting transport activity expression. This is thefirst direct demonstration of regulation of a neurotrans- mammalian central nervous system, is typically four orders

of magnitude higher in the nerve terminals than in the cleftmitter or amino acid transporter by phosphorylation. Thenature of the upstream event that stimulates glutamate (estimations of 10 mM in neurons, low millimolar range in

glial cells and submicromolar to low micromolar range intransport via EAAT2 through protein kinase C is at presentunknown. The authors hypothesize that elevation of the the glial extracellular fluid; Refs. 40, 74, 262, 458); therefore,

energy input is required. The Na/-K/-ATPase generates anextracellular glutamate concentration would stimulateNMDA receptors in astrocytic processes, resulting in acti- inwardly directed electrochemichal sodium gradient that

drives uphill the the sodium- and potassium-coupled gluta-vation of the phosphatidylinositol cycle and protein ki-nase C activation, and therefore in a more efficient clear- mate transport in neurons and glial cells (210, 262, 394).

This role of glial and neuronal glutamate transport, in main-ance of the extracellular glutamate. To our knowledge,neither this hypothesis nor the mechanism of EAAT2 stim- taining a low extracellular neurotransmitter concentration

(õ1 mM), has been postulated to be critical to protectulation has been addressed experimentally.Conformational changes of EAAT2 have been re- against exocitotoxic cell damage (63). Glutamate transport

blockers, which are nonselective for isoforms and the differ-vealed through its transport cycle (179, 266, 583). Sodium-dependent transient currents of the expressed EAAT2 ent transport activities detected in brain (454), raise extra-

cellular glutamate, alter postsynaptic potentials, and resulttransporter suggested conformational changes associatedwith binding of sodium (266, 583). More directly, limited in neurotoxicity both in vitro (33, 226, 359, 453, 460, 470)

and in vivo (326, 402, 403). This effect is blocked by non-proteolysis studies demonstrated conformational changesof purified EAAT2 associated with glutamate and sodium, NMDA glutamate receptor antagonists (459, 460) and is


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most probably because of the excessive calcium influx resulted in the identification of EAAT2 (120, 121, 424),and immunoprecipitation studies suggest that EAAT2 iso-through NMDA receptor channels (for review, see Refs. 205,

252, 394). form is the most prevalent glutamate transporter in brain(199). The brain phenotype of knockout EAAT2 mice andKnockout studies of EAAT glutamate transporter iso-

forms (416, 458, 544) revealed that the glial transporters the very low residual glutamate transport activity in corti-cal crude synaptosomes from these mice (544) have con-(EAAT2 and EAAT1) rather than the neuronal transporter

EAAT3 control the extracellular glutamate levels in brain. firmed this suggestion. In agreement with this, in the spo-radic form of amyotrophic lateral sclerosis (ALS), thereVery recently, studies on the null knockout mice for

EAAT2 and EAAT3 have been published (416, 544). To is a specific marked reduction (up to 95%) of the expres-sion of EAAT2 in the motor cortex and the spinal cordour knowledge, the null knockout EAAT1 mouse has not

been reported. Tanaka et al. (544) studied the knockout (463). In parallel, there is also a marked decrease in theVmax of high-affinity glutamate uptake in synaptosomesof the widely distributed astrocytic glutamate transporter

EAAT2 (also named GLT-1). These mice show lethal spon- from those brain structures and an increased cerebrospi-nal fluid concentration of L-glutamate and L-aspartate intaneous epileptic seizures, selective neuronal degenera-

tion in hippocampus, and increased susceptibility to acute ALS patients (462) (see sect. III).The above-mentioned knockout studies showed thatcortical injury. In these mice, the estimated peak concen-

tration and time course of free glutamate in the synaptic the glial transporters (EAAT1–2) are the more conspicu-ous transporters in brain, and they have a crucial rolecleft is elevated. This indicates that glial EAAT2 is an

important determinant of the clearance of free glutamate in the maintenance of the tonic extracellular glutamateconcentration. Thus the tonic increase in extracellularfrom the synaptic cleft. Thus, in the absence of EAAT2

transport activity, glutamate levels rise enough to cause glutamate because of EAAT1–2-specific partial knockoutsexplains the progressive paralysis and neurodegenerationepilepsy and cell death. In contrast to this, null knockout

EAAT3 mice, obtained by Stoffel and co-workers (416) in these rats (458). As discussed by Rothstein and Kuncl(458), glial cells have a considerably lower estimated in-show, in addition to the renal phenotype (see below), a

nonconspicuous brain phenotype, only characterized by tracellular glutamate concentration (in the micromolarrange) than neurons, which suggests that EAAT1–2 trans-reduced locomotor activity. Rothstein and Kuncl (458)

addressed the contribution of the three EAAT1–3 iso- porters operate far from equilibrium, most probably dueto the rapid metabolization to glutamine by glutamine syn-forms described in rat to the maintenance of global extra-

cellular glutamate concentrations in the cerebrospinal thetase, which is absent in neurons. Therefore, in additionto its larger expression, the operation of EAAT1–2 farfluid, as well as the histological and behavioral conse-

quences of their specific partial knockouts (in vitro and from equilibrium may explain why the phenotype ob-tained after total or partial knockout of EAAT1–2 isin vivo intraventricular phosphorothioate antisense ad-

ministration). At present, the cerebellar EAAT4 isoform, clearer than that given by knockout of the EAAT3 isoform(416, 458, 544). It is worth mentioning that the proposeddescribed in humans (63) and suspected to maintain ex-

tracellular glutamate concentrations below excitotoxic lack of role for the EAAT3 transporter in the tonic extra-cellular glutamate levels (458) does not imply that thislevels, has not been isolated from rat tissues. The partial

knockout of EAAT1–3 isoforms (458) showed that both transporter has no role in excitotoxicity. Reversal of gluta-mate transport has been proposed as a mechanism ofglial transporters (EAAT1–2) contribute largely to the

maintenance of the tonic cerebrospinal glutamate concen- excitotoxicity under conditions of energy failure, as incerebral ischemia (hypoxia, stroke; Refs. 23, 246, 394).tration and that the impact of the EAAT2 isoform was

more conspicuous. In contrast, the contribution of the The nonlimiting transport flux via EAAT3 running in re-verse could produce a significant local increase in theneuronal EAAT3 is negligible. In parallel, the EAAT iso-

form-specific partial knockout showed that glial gluta- extracellular concentration of glutamate (i.e.,ú350 mM asdemonstrated in salamander retinal glia cells and EAAT3mate transport sites (EAAT1–2) are more conspicuous

than the EAAT3 transport sites (binding of radiolabeled expressed in oocytes; Refs. 63, 246).Null knockout EAAT2 mice (544) confirmed the hy-D-aspartate inhibitable by DL-threo-b-hydroxyaspartate to

membranes) in the two structures studied, striatum and pothesis (394) that astroglial glutamate transporters con-tribute to the reuptake/termination of the glutamate syn-hippocampus. This is in agreement with greater expres-

sion of these transporters in comparison with the neu- aptic transmission. Thus total loss of EAAT2 transportactivity (i.e., as in the homozygous null knockout mice),ronal isoform. The EAAT2 isoform is present in astrocytes

throughout the brain and spinal cord (121, 305, 461). In but not its partial loss (i.e., as in the heterozygous nullknockout mice or in chronic antisense administration tocomparison with EAAT isoforms 1–3, partial knockout of

EAAT2 resulted in the largest decrease in the glutamate rat brain), produces epilepsy (458, 544) and increases thetime course of free glutamate in the synaptic cleft (544).transporter sites in striatum and hippocampus (458), puri-

fication through functional reconstitution from rat brain On the other hand, Amara and Kavanaugh and co-workers


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(583) and Kanner and co-workers (120) estimated a trans- EAAT3 may cause dicarboxylic aminoaciduria, an inher-ited disease due to defective glutamate transport in kidneyport cycling time of 70–700 ms for human EAAT2 ex-

pressed in oocytes and rat purified EAAT2. This is signifi- and intestine (see sect. III). Recent results showed thatsystem X0

AG transport activity is increased (Vmax effect) bycantly slower than the 1- to 2-ms time constant of gluta-mate decay estimated in hippocampal synapses (104, 112). hypertonic stress in the bovine renal cell line NBL-1 (148).

Concomitantly, the EAAT3 transcript levels increase, sug-This suggests that glutamate diffusion and ‘‘fast’’ bindingto EAAT2 transporters (583) dominates the synaptic con- gesting that this glutamate isoform is responsible for sys-

tem X0AG in these cells, and indicating a direct effect ofcentration decay kinetics.

What is the role of the neuronal EAAT3 glutamate hypertonic stress in the expression of this transporterisoform (148). Whether hypertonic stress increasestransporter in the termination of synapsis? In contrast to

the glial glutamate transporters, the partial knockout of EAAT3 gene transcription and/or mRNA stability in thesecells has not been reported. This regulation of EAAT3EAAT3 protein produced no changes in extracellular glu-

tamate and only mild neurotoxicity and motor phenotype, might be due to a role of this glutamate transporter inglutamine metabolism and pH regulation in renal cells.but consistent epileptic seizures (458). It is believed, al-

though this is not completely clear (628), that the neuronal The physiological role of ASCT1–2 and ATBo zwitter-ionic transporters is at present unclear. It is necessary toglutamate carrier EAAT3 operates at or near equilibrium,

and its expression is confined to pre- and postsynaptic clarify whether mouse ASCT2 corresponds to humanATBo, or whether they code for different transport activi-elements (461). It is somehow expected that the partial

reduction of a plasma membrane transport activity, which ties. In addition, a more precise description of the mecha-nism of transport for these transporters is needed. If, fi-is working at equilibrium (i.e., flux through this trans-

porter does not limit the overall metabolic handling of the nally, ASCT transporters mediate concentrative uptake oftheir substrate coupled with the transmembrane gradientneuronal glutamate) and confined to the synapsis, has

little or no impact in the global extracellular glutamate of sodium and amino acids, ASCT1–2 might be assignedas variants of the almost ubiquitious ASC system. It willconcentration, as the partial knockout studies showed

(458). The epileptic phenotype of the partial knockout of be also necessary to explain the molecular basis of thehepatic ASC system, which as discussed above does notEAAT3 suggests that a moderate rise in the intrasynaptic

glutamate concentration, without global concentration appear to be represented by either one of these ASCTisoforms. Studies with anti-ASCT1–2 antibodies andchanges, may cause persistent depolarization or alteration

of the presynaptic transmitter release (458). In addition knockout experiments will be needed to estimate the roleof these transporters in the macroscopic flux of aminoto glutamatergic neurons, EAAT3 has also been located

in inhibitory GABAergic neurons, and because glutamate acids in cells expressing them.The ATBo transporter (269) might correspond to sys-is a precursor for GABA synthesis, transport via EAAT3

could have a role in GABA neurotransmission (205, 461). tem Bo, the most conspicuous sodium-dependent uptakesystem for zwitterionic amino acids. This apical transportSuperstimulation of excitatory glutamatergic neurons and

blockade of inhibitory GABAergic neurons are known to system is thought to play a major role in reabsorption ofzwitterionic amino acids in kidney and small intestine (seeproduce epilepsy (157). Unfortunately, the null knockout

EAAT3 mouse model only reproduces the locomotor, but Refs. 483, 513). Elucidation of the transport mechanismand demonstration of the apical localization of ATBo innot the epileptic, phenotype (416) of the antisense-de-

pleted EAAT3 rat model (458). It seems that overex- epithelial cells may reaffirm the assignation of ATBo assystem Bo transporter. Finally, demonstration of the re-pression of the glial glutamate transporters does not occur

in the knockout EAAT3 mice (517). These apparently con- sponsibility of ATBo in Hartnup disease, an inherited neu-tral hyperaminoaciduria (see sect. III), may confirm thattradictory results raise doubts as to the contribution of

the EAAT3 transporter to the termination of glutamate ATBo plays a role in amino acid nutrition and renal reab-sorption and system Bo activity. In contrast to this view,synapsis.

Expression of the glutamate transporter EAAT3 in the recent description of an amino acid exchange mecha-nism of transport for ATBo (162) questions the participa-GABAergic neurons (205, 461), its strong transcript ex-

pression in the small intestine, and at a lower levels in tion of this transporter in the active renal and intestinalabsorption of neutral amino acids and its role in Hartnupkidney, liver, and heart (245), suggest a metabolic role for

this transporter. In epithelial cells, system X0AG has been disease.

described mainly in the apical plasma membrane (483,D. Putative Subunits of Sodium-Independent513), and therefore, it is believed that EAAT3 mediates

Cationic and Zwitterionic Amino Acidnet absorption of glutamate and aspartate in kidney andTransportersintestine (205). This role is demonstrated by the dicarbox-

ylic aminoaciduria developed by null knockout EAAT3 The last protein family related to plasma membraneamino acid transport in mammals is composed by themice (416). In addition, this suggests that mutations in


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TABLE 8. Putative subunits of sodium-independent cationic and zwitterionic amino acid transporters

Putative Transporter Human ProteinSubunits (Gene Accession Numbers Origin of First Clones and Human Amino Acid

Name) (Origin of Human Clones) Other Names Chromosome Length Other Clones

rBAT (SLC3A1) L11696 (kidney) (45) Rat kidney (NAAT, NBAT) 2p16.3-21 (79, 685 rBAT long transcript (rabbitM95548, M95298 (kidney) (549) 616, 629) kidney) (344)

(297) Rabbit kidney (rBAT) (46) rBAT short transcriptD82326 (kidney) (367) Rat kidney (D2) (598) (rabbit kidney and OK

cells) (110, 374)4F2hc (SLC3A2) J02939, M17430, M18811, Human lymphocytes (4F2hc) 11q12-13 (174) 529 Mouse brain (413),

M21904 (lymphocytes) (435, 553) (corresponds to hematopoietic stem cells(327, 435, 554) CD98) (592), and rat glioma

J03569 (fibroblasts) (327) cells (69)

Accession numbers for human rBAT and 4F2hc cDNAs are indicated. Alternative names for other cDNA are shown. Reference numbers aregiven in parentheses.

protein rBAT and the heavy chain of the cell surface anti- only 76% identical to the rat and mouse proteins (69).More recently, using an antibody that induces apoptosisgen 4F2 (4F2hc) (see Table 8). Amino acid transport ex-

pression in Xenopus oocytes was used independently in in hematopoietic progenitor cells and homotypic aggrega-tion of lymphoid progenitor cells as a screening tool inthree labs to clone cDNA of a putative transporter from

rabbit, rat, and human kidney; homology between these transiently transfected COS-1 cells, the mouse 4F2hc wascloned again (592). As discussed in section IID5, 4F2hcproteins is very high (Ç85% identity) (45, 46, 110, 297,

549, 598). A partial rBAT cDNA sequence from OK cells might have multiple functions.The relevance of rBAT in the reabsorption of cystinehas also been reported (374). The three labs gave different

names to these cDNA: NBAT (Udenfriend and Tate’s and dibasic amino acids in kidney and intestine has beendemonstrated by the involvement of the rBAT genegroup), D2 (Hediger’s group), and rBAT (ourselves). For

clarity, the name rBAT will be used for all these cDNA (named SLC3A1 in Gene Data Bank) in cystinuria (forrecent reviews, see Refs. 170, 408, 467). This is an inher-and proteins in this review. The cDNA of human 4F2hc

was cloned using a monoclonal antibody designed against ited aminoaciduria due to defective renal and intestinalreabsorption of cystine and dibasic amino acids; the poora cell surface antigen from lymphoblastoid cells (327, 435,

553), and its mouse counterpart was identified by homol- solubility of cystine causes the formation of renal cystinecalculi (351, 487). Surprisingly, rBAT and also 4F2hc areogy (413). The biological role of this antigen was un-

known. The deduced rBAT protein amino acid sequences not very hydrophobic, and they seem to be unable toprovide an aqueous translocation pathway in the plasmahave Ç30% identity (Ç50% similarity) with the heavy

chain of the cell surface antigens 4F2 (4F2hc) (69, 413, membrane. This prompted the hypothesis that rBAT and4F2hc are subunits or modulators of the corresponding435, 553). Figure 7 shows the sequence homology between

the human rBAT and 4F2hc proteins, and in Figure 8, the amino acid transporters. In this sense, it has been sug-gested or demonstrated that there is an association ofamino acid residues conserved in all rBAT and 4F2hc

proteins known are indicated. Consistent with rBAT and rBAT and 4F2hc, respectively, with a corresponding lightsubunit of Ç40 kDa. Here attention is focused on the4F2hc being members of the same family, within the open

reading frame of human rBAT and 4F2hc, introns 1 and hypothesis that both rBAT and 4F2hc are subunits of theactual amino acid transporters corresponding to system2 have identical locations, intron 3 in 4F2hc corresponds

to intron 4 in rBAT, and intron 8 in 4F2hc to intron 9 in bo,/-like and y/L-like. Structural and functional evidencein favor of this is discussed. The role of the rBAT generBAT (174, 434, 431) (see Fig. 8). Given the homology

between rBAT and 4F2hc, cRNA from 4F2hc was tested in cystinuria is described in section III.in oocytes for expression of amino acid transport activity.Expression of 4F2hc resulted in an amino acid transport 1. Tissue expressionactivity (system y/L- like) different from that elicited byrBAT (system bo,/-like) (42, 599) (see Table 9). Interest- The rBAT mRNA is expressed in the kidney and theingly, expression cloning in oocytes after a zwitterionic mucosa of the small intestine (45–47, 297, 598, 617). Con-amino acid transport signal (68) resulted in the isolation sistent with this, hybrid depletion with rBAT antisenseof rat 4F2hc (named lLAT in this study for linked to L oligonucleotides blocks expression of system bo,/-like byamino acid transport) (69). It is worth mentioning that rat renal and intestinal poly(A)/ RNA in oocytes (45, 338,and mouse 4F2hc proteins are very similar (91% amino 598). Northern blot analysis of human, rat, and rabbit

renal and intestinal RNA revealed two rBAT transcripts:acid sequence identity), whereas the human protein is


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FIG. 7. Amino acid sequence comparison of humanrBAT and 4F2hc proteins. Thick horizontal line over se-quences indicates hydrophobic segment that correspondsto first putative TM domain, whereas thin horizontal linesindicate amphipathic TM domains II-IV proposed by Tateand co-workers (378). Solid frame box in gray indicatesresidues that resemble catalytic site of homologous glycos-idases. Here, arrows indicate position of proposed cata-lytic residue (aspartate or glutamate) of these glucosi-dases; this residue is substituted by arginine in human4F2hc and by asparagine in rabbit rBAT (42). In gray boxesare indicated amino acid residues present in human rBATand 4F2hc proteins. Four dash frame boxes indicate seg-ments of 12–17 amino acid residues with high homologybetween rBAT and 4F2hc proteins. Solid frame boxes indi-cate potential N-glycosylation sites, 6 for rBAT and 2 for4F2hc. Dash indicates gaps for sequence multialignmentobtained with all known rBAT and 4F2hc sequences withClustal Sequence Alignment from Baylor College of Medi-cine.

Ç2.3 kb (which corresponds to the above-mentioned immunoreactivity in hypothalamus is intracellular, and itcDNA) and Ç4 kb. A cDNA corresponding to the long is not located in the plasma membrane as in kidney andrBAT transcript was identified by expression cloning in intestine (212). One antibody directed against a peptideoocytes and represents an alternative polyadenylation of of the rBAT sequence labeled intracellular structures ofthe same gene (344). In situ hybridization and immunolo- magnocellular neurons of the supraoptic and paraventric-calization studies have demonstrated that rBAT localizes ular nuclei.to the microvilli of the small intestinal mucosa and the In contrast to rBAT, 4F2hc mRNA is almost ubiquitousepithelial cells of the proximal straight tubules of the in mouse tissues, with a higher expression level in testis,nephron (159, 248, 422). Interestingly, the expression of lung, kidney, brain, and spleen and without a clear patternrBAT is developmentally regulated in rat kidney; rBAT of developmental regulation (413). Studies previous even totranscripts appear after birth, and the onset of the protein the cloning of 4F2hc showed that this protein is inducedexpression coincides with postnatal nephron maturation after activation of human and mouse lymphocytes (reviewed(159). Clear rBAT transcripts are also visible in human in Ref. 413; see sect. IID5). In fibroblasts (NIH 3T3 andpancreas; the significance of rBAT expression in pancreas BALB/c 3T3 cells), 4F2hc expression is induced during cellis unknown (45). In addition to kidney and intestine, brain activation and maintained high throughout the cell cycle intissues show a transcript of Ç5 kb that hybridizes with exponentially growing cells (413). This suggests that 4F2hcrBAT cDNA probes (45, 46, 617). This long transcript is plays a role in proliferating and quiescent cells. The aminoalmost ubiquitous, but with a substantially lower abun- acid transport activities associated with 4F2hc, as describeddance in tissues other than brain (45). The RNA protection here, may be relevant for both situations.assay studies and Western blot analysis with some but

2. Transport propertiesnot all anti-rBAT peptide antibodies suggested that thislong transcript corresponds to the expression of a gene The characteristics of the amino acid transport activ-

ity associated with rBAT and 4F2hc expression have beenthat is homologous to rBAT (422, 617). Moreover, rBAT


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FIG. 8. Cystinuria-specific mutants in 4 TM domain topology model of human rBAT protein. This model has beenproposed by Tate and co-workers (378) (see text for details). Amino acid residues conserved in all rBAT proteins(including OK cell rBAT protein fragment corresponding to human residues 373–593) are indicated in gray circles andthose present also in 4F2hc proteins in white letters on a black circle. Notice that cysteine residue 114, after first TMdomain, is conserved in all these proteins. Twenty-one cystinuria-specific mutations are indicated by arrows. Numbershows amino acid residue involved. For 15 missense mutations, substitution amino acid is indicated. Most of thesemutations occur in conserved amino acid residues, with exception of R181Q, T652R, and F648S. ns, fs, and sm denotenonsense, frame shift, and splice mutations, respectively. In addition, four large deletion mutations have been describedwith the following approximate boundaries: 114–1306, 198–1575, 430–765, and a mutation affecting exons V to X;position 1 corresponds to 5*-position of first ATG codon. All mutations referred to here have been reviewed in References170 and 408. Four of these mutations (E268K, T341A, M467T, and M467K; amino acid residues indicated by an square)have been analyzed in oocytes and show defective transport expression (78, 91, 366). Six potential N-glycosylation sites(Y) are indicated in first and second putative extracellular loops. Drawing of extracellular and intracellular loops andNH2 and COOH terminals do not indicate any type of structure. Exon-intron boundaries of human rBAT are shown asreported in References 434 and 431. Boundaries conserved in open reading frame of human 4F2hc (174) are indicatedby an asterisk.

studied mainly in oocytes. rBAT induces, through the oo- oocytes, at least in sodium-free medium (see below) (46),which is not present in stage VI oocytes (42, 46, 354). Thiscyte plasma membrane, transport of cystine (up to ú100-

fold over background) and dibasic and zwitterionic amino transport activity is sodium independent, and it is verysimilar to the amino acid transport system bo,/ defined byacids (up to 50-fold over background). This is a high-

affinity transport with Km values in the micromolar range Van Winkle et al. (577) in mouse blastocysts, as a sodium-independent high-affinity system for dibasic and zwitter-for amino acids such as L-cystine, L-arginine, L-lysine, L-

ornithine, L-leucine, and L-histidine. Kinetic and cross-in- ionic amino acids. In contrast to the transport systemassociated with rBAT, the blastocyst bo,/ system does nothibition studies offered convincing evidence that rBAT

induces a single amino acid transport system in Xenopus transport L-cystine (L. J. Van Winkle, personal communi-


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TABLE 9. Tissue distribution and transport characteristics of expressed rBAT and 4F2hc proteins

Transporter Tissue Distribution Substrates Cotransported AccumulationSubunit (Transcript Size) Expression System (Km, mM) Ligands Amino Acid Exchange of Substrates

rBAT Epithelial cells (mainly Oocytesc System bo,/-like* None (75, 110) Homo- and heteroexchange Ç50-fold (90)kidney and small Partial knockout substrates of aa/ and aao,g

intestine)a (Ç2.4 kb, in OK cells (Ç50)e Favored exchange (90): 1Ç4 kb) (374) L-Cystine, aa/ aa/ (influx)/1 aao (efflux)

and some aao

(e.g., L-Leu)4F2hc Ubiquitousb (Ç2 kb) Oocytesd System y/L-like* Na/ with aao,f Favored exchange (90): 1 Ç30-fold (90)

Antisense in substrates aao plus Na/ (influx)/1blood cells (Ç50)f: aa/ aa/ (efflux)(88) and aao plus

Na/

Transcript sizes of rBAT are similar in human, rat, and rabbit tissues, and 4F2hc transcript size is similar in human and mouse tissues.* Amino acid transport activity of system bo,/-like for rBAT expression in oocytes and OK cells, and of system y/L-like for 4F2hc expression inoocytes described by Tate’s, Hediger’s, Ganapathy’s and our groups (see references below) are shown. Others proposed induction of sodium-dependent histidine transport in rBAT-injected oocytes (4) and sodium-independent transport for both dibasic and zwitterionic amino acids (69)(see text for details). Values of substrate accumulation in oocytes were obtained at 50 mM radiolabeled susbtrates. aao, Zwitterionic amino acids;aa/, dibasic amino acids. References are as follows: a) 45, 46, 297, 344, 598, 617; b) 413, 435, 553; c) 3, 45, 46, 75, 78, 90, 91, 109, 110, 297, 338,344, 374, 367, 549, 598; d) 42, 69, 90, 142, 146, 338, 599; e) 4, 45, 46, 75, 91, 110, 297, 338, 344, 367, 374, 598, 617; f) 42, 142, 146, 599; g) 3, 75, 90,91, 109, 110. Other references are given in parentheses.

cation). For this reason, we named our human and rabbit duced in rBAT-expressing oocytes remains to be demon-strated.cDNA clones rBAT, as an acronym for ‘‘related to bo,/

amino acid transporter’’). Further characterization of the In contrast to rBAT, the cRNA of human 4F2hc in-duces an amino acid transport activity (e.g., up to 10-foldrBAT/system bo,/-like transport activity showed that it

was independent of external potassium and chloride (75), over background for radiolabeled L-arginine), which issodium independent with high affinity (micromolar range)changes in the external pH (Palacın, unpublished data),

and internal ATP (110). Cystine and dibasic and zwitter- for L-dibasic amino acids, but with high affinity for L-zwitterionic amino acids only in the presence of sodium;ionic amino acid transport with the characteristics of sys-

tem bo,/-like have been described in renal and intestinal in the absence of sodium, the affinity for L-zwitterionicamino acids is dramatically reduced (46, 599). This trans-plasma membrane preparations (see sect. IID5).

Ahmed et al. (4) and Taylor and co-workers (420) port activity, which does not transport L-cystine, is verysimilar to the system y/L, initially described in humanpropose that the expression of rBAT induces several

amino acid transport systems: 1) a NEM-resistant sodium- erythrocytes by Deves et al. (127), and later describedin brush-border membrane vesicles from human placentaindependent transport for cationic and zwitterionic amino

acids, equivalent to system bo,/-like (R. Estevez and M. (135); a recent review (126) describes the transport char-acteristics of system y/L. In the same line, Ganapathy andPalacın, unpublished data), and in brush-border mem-

brane preparations of chicken jejunum (560); 2) sodium- co-workers (146) have shown that poly(A)/ RNA from ahuman choriocarcinoma cell line expresses y/L transportindependent transport activities (perhaps two), which are

sensitive to NEM treatment, and with overlapping spe- activity in oocytes that is hybrid depleted by 4F2hc anti-sense oligonucleotides. Very recently, similar data havecificties for cationic and zwitterionic amino acids; and 3)

a sodium-dependent transport for L-histidine, which has been obtained with rat lung poly(A)/ RNA (142). In con-trast to this, Broer et al. (69) showed a clear induction bya pH dependence compatible with the transport of this

substrate in the nonprotonated form. Then, either as a rat 4F2hc in oocytes of a high-affinity uptake for cationicand zwitterionic amino acids, both in the absence of so-consequence of the overexpression of rBAT in oocytes or

reflecting a true mechanism of activation, rBAT induces dium. The induced transport activity has combined char-acteristics of system bo,/-like and L-like, but not of systemseveral amino acid transport activities in the oocyte (see

sect. IID4). The fact that partial knockout of rBAT in OK y/L-like (see sect. IID4).The fact that mutations in the rBAT gene cause cysti-cells results in a specific, partial decrease in the apical

system bo,/-like activity (374), and the finding that muta- nuria (see sect. III), a defect in the renal and intestinalreabsorption of cystine and dibasic amino acids, raisedtions in the rBAT gene cause cystinuria (for review, see

Refs. 170, 408, 467) demonstrate the role of rBAT in the an important question: how does a sodium-, potassium-,proton-, and ATP-independent transporter such as thehigh-affinity reabsorption system of cystine (system bo,/-

like) (see sect. IID5). In this context, the physiological bo,/-like system associated with rBAT participate in anactive process, like the reabsorption of cystine and dibasicrelevance of the other amino acid transport activities in-


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amino acids? The answer came from the study of the vice versa), the current elicited by the substrates in oo-cytes expressing rBAT is maximal, and with a directionelectrical activity of system bo,/-like. Busch et al. (75)

studied this activity of the system bo,/-like expressed by corresponding to the exchange. At a defined membranepotential (e.g., 050 mV), the exchange L-arginine (in-rBAT in oocytes; as expected, in oocytes expressing rBAT,

but not in control oocytes injected with water, the pres- flux)/L-leucine (efflux) is favored versus the reverse ex-change. This demonstrated that the exchange of sub-ence of L-arginine in the medium produces an inward posi-

tive current, most probably due to the positive charge of strates via rBAT/system bo,/-like is the only electric ac-tivity of this transporter, in agreement with previousarginine at neutral pH. Surprisingly, exposure of rBAT-

expressing oocytes to L-leucine produced an outward pos- data obtained with the cut-open oocyte model (110). Inaddition, both influx and efflux require substrates onitive current through the plasma membrane of the oocyte.

The participation of ions (e.g., Na/, K/, Cl0) in these cur- both sides; no electric activity is evoked by substratesin rBAT-expressing cut-open oocytes, which is entirelyrents was ruled out. These results prompted the hypothe-

sis that the bo,/-like/rBAT transporter exchanges amino due to heteroexchange of substrates, if no substratesare present on the trans-side (109, 110).acids through the plasma membrane; the outward positive

current produced by zwitterionic amino acids (e.g., L-leu- 5) In conditions of homogeneous exchange, eitherhomo- or heteroexchange, radiolabeled substrate effluxcine) would be due to the concomitant exit of dibasic

amino acids from the oocyte. This was demonstrated in equals influx. This together with estimations of Hill coef-ficients of approximately one both in radiolabeled andseveral laboratories by testing the dependence of amino

acid efflux from oocytes expressing rBAT on the external electric amino acid transport measurements (45, 47, 75,90) indicates a stoichiometry of exchange of one aminoamino acids; the efflux of L-[3H]arginine and L-[3H]leucine

is totally dependent of the presence of amino acids in the acid (influx)/one amino acid (efflux).The tightness of the obligatory exchange mechanismmedium (3, 90, 110). In fact, Coady et al. (110) have iso-

lated a renal rabbit rBAT cDNA by expression of the elec- of rBAT/system bo,/-like is at present unknown. The factthat cationic amino acid-evoked currents occur in rBAT-tric activity of system bo,/-like/rBAT in oocytes. Additional

data confirmed that system bo,/-like is an obligatory ex- expressing cut-open oocytes only when zwitterionicamino acids are present on the trans-side favors a verychanger, which acts as a tertiary active transporter (90).

1) Only the system bo,/-like substrates elicited efflux tight coupling of exchange (109). With the assumption ofa theoretical absolute requirement of the transporter tovia system rBAT/bo,/-like in oocytes.

2) The exchange mechanism is able to accumulate be occupied by a substrate at either side (intra- or extra-cellularly) to translocate (this is thermodynamically im-amino acid substrates in oocytes expressing rBAT; Ç50-

fold intracellular accumulation of 50 mM extracellular ra- possible) the amino acid, accumulation curves via rBAT/system bo,/-like have been modeled (90). If the transportdiolabeled L-arginine, L-leucine, or L-cystine. This level of

accumulation is not due to metabolism of the radiolabeled model assumes that the velocity constants of transloca-tion of the empty (no substrate bound at either side) trans-substrate in the oocytes, and it is significantly higher than

that obtained in noninjected oocytes, or in oocytes ex- porter is Ç30-fold lower than for the amino acid-trans-porter complex, the experimental accumulation curvespressing the cationic amino acid transporter CAT1 (sys-

tem y/ activity, which shows trans-stimulation but is not may still be reproduced by the model (J. L. Gelpı and M.Palacın, unpublished data); the sensitivity of the transportan exchanger; see sect. IIA2).

3) The active transport due to rBAT/system bo,/-like studies of radiolabeled substrates in oocytes expressingrBAT precludes a more precise determination. The mech-expression in oocytes has a limit of accumulation, which

coincides with the amount of intracellular free amino acid anism of exchange of rBAT/system bo,/-like (sequential orconcerted substrate binding at both sides) is at presentsubstrates in the oocyte (these cells contain a very high

intracellular concentration of amino acids that has been unknown. Interestingly, when the analog aminoisobutyricacid is used as a substrate, the amino acid exchange viaestimated to be Ç2,500 mM zwitterionic amino acids and

750 mM dibasic amino acids; Ref. 550). rBAT/system bo,/-like shows a variable stoichiometry ofexchange: the aminoisobutyric acid-induced currents (i.e.,4) As a consequence of this, prolonged incubations

of the rBAT-expressing oocytes in the presence of an efflux of the positively charged cationic amino acid sub-strates) is higher than the concomitant aminoisobutyricrBAT/system bo,/-like substrate results in the complete

exchange of this amino acid within the oocyte. In this acid radiolabeled transport flux (109). This suggests thataminoisobutyric acid ‘‘locks’’ the transporter in a confor-situation (homogeneous exchange), and in voltage-

clamp conditions, when homoexchange is forced (e.g., mation that enables free translocation of the transporterin a fraction of transport cycles. Accumulation studiesL-arginine influx and efflux or L-leucine influx and ef-

flux), the electric activity of rBAT/system bo,/-like disap- with aminoisobutyric acid (predicted to be lower thanwith physiological substrates) have not been reported.pears. In contrast, when homogeneous heteroexchange

is forced (e.g., L-arginine influx and L-leucine efflux, or The exchange mechanism of rBAT/system bo,/-like is


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not an oocyte artifact. It has also been shown to occur membrane preparations in conditions in which othertransport activities are blocked (i.e., NEM-treated vesiclesin renal cells that naturally express rBAT; the gene is

expressed, and the system bo,/-like is present in the ‘‘renal to inhibit system y/ and in the presence of intravesicularBCH, a system L inhibitor)?proximal tubular’’ OK cell line (374). 1) In the apical pole,

the transport of cystine and most of the L-arginine trans-port (Ç80%) has the substrate specificity of system bo,/- 3. Protein structurelike. 2) The substrate efflux via system bo,/-like showscomplete dependence on external amino acid substrates. rBAT and 4F2hc proteins have no membrane leader

sequence, similar hydrophobicity plots (reviewed in Ref.3) This transport activity is due to the expression of therBAT gene; stable transfection with antisense rBAT se- 407), and four regions (12–17 amino acid residues long)

highly conserved (67–80% identity) (see Fig. 7). Both pro-quences results in the partial and specific loss of systembo,/-like activity. This demonstrated that the exchange teins also have a domain with significant homology with

a protein family of prokaryotes and insect a-amylases andmechanism for rBAT/system bo,/-like also occurs in epi-thelial renal cells. This is in full agreement with heteroex- a-glucosidases (42, 46, 598). Interestingly, the catalytic

site of these glucosidases is not totally conserved in rabbitchange of cystine and dibasic amino acids observed inrenal brush-border membrane vesicles (352). The sodium- rBAT or human 4F2hc; this is consistent with the fact that

expression of rBAT in oocytes does not show a-amylasedependent L-histidine transport induced by rBAT in oo-cytes (4) is currently being studied in OK cells to assess or maltase activity (598). In contrast to the well-known

membrane multispanning structure of membrane trans-the physiological relevance of this induction.Interestingly, the y/L-like transport activity induced porters of substrates of polar nature (607), rBAT and 4F2

(4F2hc) are less hydrophobic and contain, depending onby 4F2hc in oocytes also behaves as an exchanger (90).1) Efflux of radiolabeled L-arginine via 4F2hc/system y/L- prognosis based on hydrophobicity algorithms, a single

TM domain (i.e., a type II membrane glycoprotein; Refs.like requires the extracellular presence of its substrates(e.g., cationic amino acids in sodium-free medium or zwit- 46, 435, 553, 598) or four TM domains, with intracellular

NH2 and COOH termini; Ref. 549); the more NH2-terminalterionic amino acids in sodium-containing medium). 2)As a consequence of this exchange mechanism, oocytes hypothetical TM domain is the only one showing a clear

prognosis as a TM domain (see Fig. 7). Surprisingly, theseexpressing 4F2hc are able to accumulate system y/L-likesubstrates at higher levels than noninjected oocytes or structures induce amino acid transport activity via system

bo,/-like and y/L-like in Xenopus oocytes, respectively,CAT1/system y/-injected oocytes. 3) The exchange ofamino acids via 4F2hc/system y/L-like is asymmetric. Ef- and the involvement of rBAT in cystinuria demonstrates

a role for rBAT in renal and intestinal reabsorption offlux of radiolabeled L-leucine is not observed in oocytesexpressing 4F2hc even in the presence of extracellular amino acids. The apparent inability of these proteins to

provide an aqueous translocation pathway in the plasmasubstrates; this is interpreted as showing that the interac-tion of zwitterionic amino acids with 4F2hc/system y/L- membrane, due to their low hydrophobicity, prompted the

hypothesis that they may be modulators of transporterslike at the low intracellular sodium concentration is veryweak and therefore not visible in radiolabeled uptake with a heteromeric structure (42, 46, 598).

Biochemical and immunochemical studies have dem-studies. This suggests that exchange via 4F2hc/systemy/L-like favors the efflux of cationic amino acids and so- onstrated that rBAT and 4F2hc are integral membrane N-

glycoproteins. The experimental evidence for rBAT candium-dependent influx of zwitterionic amino acids. Theerythrocyte/placental system y/L shows marked trans- be summarized as follows: 1) in vitro translation. Addition

of microsomes to the reticulocyte translation system in-stimulation, compatible with a ratio of velocity constantsfor the translocation of the occupied and empty carrier creases (õ20 kDa) the molecular mass of the protein

product synthesized from rBAT cRNA (344, 598). 2) rBATof at least 25 (14, 127, 135). It is therefore also possiblethat system y/L may indeed be acting as an exchanger. is expressed in oocytes. The protein product (Ç90 kDa)

from rBAT cRNA in oocytes, shown by metabolic labelingIn this sense, as discussed previously for the modeling ofthe accumulation of substrates in oocytes via rBAT/sys- with [35S]methionine, is an integral N-glycoprotein. Thus

the product is not solubilized from oocyte membranes bytem bo,/-like, the level of trans-stimulation of system y/Lwould allow transient accumulation of substrates similar sodium carbonate treatment. The treatment of the oocytes

with tunicamycin reduces the size of the protein to Ç72to those observed in 4F2hc-expressing oocytes (90). Itwould be interesting to discern whether the asymmetric kDa, compatible with the mass of the deduced protein

from the cDNA (Mr õ79 1 103) (45). 3) Studies with theexchange (efflux of cationic amino acids/influx of zwitter-ionic amino acids plus Na/) observed via system y/L- native protein have been done. Western blot analysis using

specific anti-rBAT antibodies revealed a protein band oflike activity in 4F2hc-injected oocytes also happens in theerythrocyte/placental y/L system. What is the nature of 90–95 kDa in membrane preparations from kidney and

mucosa from the small intestine (159, 379). The size ofthe sodium dependence of L-leucine efflux from plasma


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this band is reduced to Ç72 kDa after endoglycosidase F 4. Structure-function relationship

treatment of renal brush-border membranes (J. ChillaronThe studies on structure-function relationship onand M. Palacın, unpublished data). Because of the lack of

rBAT/system bo,/-like and 4F2hc/system y/L-like areleader peptide and because the N-glycosylation sites arescarce, and limited to, the defect in four cystinuria-spe-toward the COOH terminus from the location of the mostcific rBAT mutants (78, 91, 366), the effect of a COOH-evident putative transmembrane domain (see Fig. 7), itterminal deletion of rBAT (367), and indirect evidencewas proposed that rBAT and 4F2hc were type II mem-that supports the hypothesis that the functional units ofbrane glycoproteins (i.e., cytosolic NH2 terminus and ex-the systems bo,/-like and y/L-like are heterodimeric struc-tracellular COOH terminus) (46, 435, 553, 598). In con-tures of rBAT and 4F2hc with their corresponding puta-trast, Tate and co-workers (378) have proposed that rBATtive ‘‘light subunits’’ (see below; see Refs. 408, 409, 548).crosses the plasma membrane at least four times, with

Four human cystinuria-specific rBAT missense muta-the first transmembrane domain already mentioned andtions have been tested for amino acid transport expres-three additional amphipathic transmembrane domainssion in oocytes (Met467Thr and Met467Lys, Refs. 78 and(Fig. 8). This is based on studies of limited proteolysis91; E268K and T341A, Ref. 366) (see location of theseand peptide-specific antibody detection of permeabilizedamino acid residues in Fig. 8). All four show partiallycells expressing the rBAT protein (378). These highly in-defective amino acid transport when expressed in oo-teresting results on the rBAT protein await confirmationcytes. Expression in oocytes of Met467Thr, the most fre-with different approaches; no similar studies have beenquent cystinuria type I mutant known worldwide (it repre-conducted with 4F2hc. In any case, it seems that one orsents 26% of the type I cystinuria chromosomes explained;four transmembrane domains are not enough to conformsee sect. III), and the mutant Met467Lys results in a re-a polar pore for the movement of amino acids throughduced Vmax of the induced system bo,/-like, without sub-the plasma membrane.stantial effect on the apparent Km; this is not due to de-rBAT and 4F2hc might be components of heteromericfects in the synthesis or degradation of the transporteramino acid transporters (42, 46, 599): rBAT and 4F2hc(78, 91). A deeper study on the transport defect associatedmay be ‘‘activators’’ of silent bo,/-like and y/-like trans-with Met467Thr and Met467Lys mutants revealed aporters of the oocyte, respectively. A possible mechanismplasma membrane trafficking defect. These mutants ex-for this activation could be the constitution of holotrans-press only an endoglycosidase H-sensitive protein bandporters with subunits present in the Xenopus oocytes.in the oocytes, and the protein reaches the oocyte plasmaThis hypothetical mechanism would be similar to the acti-membrane slowly and inefficiently, as revealed by surfacevation of the oocyte a-catalytic subunits of the Na/-K/-biotinylation studies (91). Long oocyte expression periodsATPase by the expression of foreign b-subunits of the(ú3 days after injection) and injection of oversaturatingNa/-K/-ATPase (166); a similar mechanism has been de-amounts of mutant rBAT cRNA result in total (Met467Thr)scribed for multimeric channels (32, 206, 469). In thisor partial (°20% activity of the wild type for Met467Lys)sense it is very interesting that the cell surface antigenrecovery of the induced amino acid transport (91); it is4F2 is a heterodimer (Ç125 kDa) composed of a heavyinterpreted that these conditions overcome the proteinchain of 85 kDa (4F2hc, i.e., the homologous protein toquality control machinery of the oocyte. Interestingly,rBAT) and a light chain of 40 kDa linked by disulfidewhen the amino acid transport activity induced bybridges (204, 209). Unfortunately, this light subunit evi-Met467Thr mutant is recovered, the amount of Met467Thrdenced by 125I labeling and immunoprecipitation has noton the oocyte surface is only õ10% of the correspondingbeen microsequenced or cloned. In a similar way, Wangwild-type protein; this suggests that an oocyte ‘‘factor’’and Tate (591) have reported the presence of these com-limits the expression of system bo,/-like activity whenplexes in brush-border preparations from kidney and in-oversaturating amounts of rBAT cRNA are expressed (91).testine. In our hands, renal rBAT is immunodetected in

In an interesting study, Miyamoto et al. (367) showedWestern blot studies in nonreducing conditions as com-that a COOH-terminus deletion (D511–685) on humanplexes of Ç240 and Ç125 kDa; in two-dimensional gelsrBAT, which eliminates the fourth putative TM domain as(first in nonreducing conditions, then in reducing condi-well as the fourth segment of high homology betweentions), the 240-kDa and the 125-kDa bands contribute torBAT and 4F2hc (see Figs. 7 and 8), induces in oocytesthe Ç90 kDa seen in reducing conditions (408). Interest-a decreased amino acid transport activity (radiolabeledingly, in membranes obtained in the presence of NEMamino acid transport studies) that resembles that offrom oocytes expressing rBAT, complexes similar in size4F2hc/system y/L-like (i.e., sodium-independent transportto those observed in kidney have been reported (591). Allof dibasic amino acids and sodium-dependent transportthis suggests that similarly to 4F2 antigen, rBAT forms aof zwitterionic amino acids); expression of longer dele-heterodimeric structure (125 kDa) of a ‘‘heavy chain’’tions in the COOH terminus of rBAT renders no transport(Ç90 kDa) linked by disulfide bridges to a putative ‘‘light

chain’’ of 40–50 kDa. function in oocytes. This suggests that rBAT and 4F2hc


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are modulators or subunits of the complete transporters, tional unit of these transporters is a heterodimer (rBATor 4F2hc plus the corresponding putative light subunit).in which the substrate specificity of systems bo,/-like and

y/L-like resides. In addition, this study suggests that the As already mentioned, two labs recently showed induc-tion of additional amino acid transport activities withCOOH terminus is relevant for the interaction with the

putative transporter or subunit. There is one concern in rBAT and 4F2hc. Ahmed et al. (4) showed sodium-depen-dent histidine uptake induced by rBAT in oocytes, in addi-this interpretation of these results. D511–685 rBAT ex-

presses substantial substrate-evoked currents (15–20 nA tion to the induction of system bo,/-like activity. Broer etal. (69) showed induction by rat 4F2hc in oocytes of a typeby 50 mM L-arginine or L-leucine at050 mV) in the oocytes

(367). In contrast, substrate-evoked currents by 4F2hc in of system L-like transport activity (sodium-independenttransport for cationic and some zwitterionic amino acids).oocytes are very small (°1 nA by 50 mM L-arginine or L-

leucine at 050 mV) (90). In agreement with this, placental This is in contrast to others who showed induction ofsystem y/L-like activity (sodium-independent transporty/L activity is largely insensitive to membrane potential

(135). This is interpreted as the reflection of the cotrans- for cationic and sodium-dependent transport for somezwitterionic amino acids) by both human and rat 4F2hcport of sodium with zwitterionic amino acids in exchange

with cationic amino acids via y/L, resulting in no electric (46, 142, 146, 599). The physiological relevance of theinduction of sodium-dependent uptake by rBAT and a sys-activity (90). At present, there is no explanation as to

why D511–685 rBAT induces in oocytes an amino acid tem L-like is at present unknown. Knockout studies simi-lar to that reported for rBAT in OK cells (374) are neces-transport activity that is identical to 4F2hc/system y/L-

like when transport is measured with radiolabeled sub- sary to assess the physiological relevance of these aminoacid inductions. Nevertheless, this opens the possibilitystrates but differs in its electrical activity.

Additional evidence also points to rBAT and 4F2hc that several putative light subunits in combination withrBAT and 4F2hc, or unidentified homologous proteins,as modulators or subunits of the amino acid transporter.

Transient expression of rBAT in COS cells resulted in the constitute different amino acid transport systems. If thehypothesis of the heterodimeric holotransporters forproduction of a glycosylated rBAT form that either does

not reach the plasma membrane (our experiments; Ref. rBAT and 4F2 is valid, the amino acid transport systemsbo,/-like and y/L-like will be the first examples of hetero-408) or does so (Tate’s group experiments; Ref. 378) but,

in both cases, with no amino acid transport expression. meric transporters for organic substrates in mammals.Knowledge of the structure-function relationship of rBATInterestingly, in nonreducing conditions, the renal and in-

testinal characteristic Ç125 kDa rBAT complex is not and 4F2hc urgently needs the isolation and cloning of thelight subunit of 4F2 and the putative light subunit of rBAT.present; it might be that the putative ‘‘light subunit’’ of

rBAT is not expressed in COS cells, precluding transport Purification of the Ç125-kDa rBAT complex by classicalbiochemical ways and coexpression cloning strategies areexpression (408). Recently, we obtained additional func-

tional evidence for the need of the putative light subunit currently in progress in several labs in an attempt to iden-tify these subunits.in the 4F2hc-induced expression of system y/L-like (142):

1) there is dissociation between oocyte surface 4F2hcprotein and induced amino acid transport activity (satura- 5. Physiological role of rBAT and 4F2hction of induced amino acid transport occurs at very lowamounts of injected cRNA, 0.01–0.1 ng 4F2hc cRNA/oo- Our knowledge of the physiological role of rBAT is

clearly greater than that of 4F2hc (see Ref. 408 for a recentcyte); expression of larger amounts of cRNA results inmore 4F2hc on the surface without increment in the in- review). The involvement of rBAT in classic cystinuria

demonstrates the role of rBAT in the renal and intestinalduced uptake. 2) In addition, there is coexpression ofsystem y/L-like activity upon injection of saturating doses reabsorption of cystine and dibasic amino acids. Because

of the cellular localization of the rBAT protein and itsof 4F2hc plus rat lung mRNA or plus rat lung size-fraction-ated mRNA; 4F2hc is necessary for this coexpression mechanism of exchange (tertiary) active transport (see

above), we proposed a model for the physiological rolesince 4F2hc antisense oligonucleotides specifically hy-brid-deplete the coexpression of system y/L-like activity of transporter bo,/-like in the renal reabsorption of cystine

and dibasic amino acids (90). In this model, the function(Estevez and Palacın, unpublished data).In summary, the studies with the Met467Thr rBAT of the transporter is directed toward apical reabsorption

of cystine and dibasic amino acids, dissipating the intra-mutant, the COOH-terminal deletion of rBAT and thecoexpression of 4F2hc and rat-lung mRNA strongly sug- cellular gradient of zwitterionic amino acids. The negative

membrane potential and the intracellular reduction of cys-gest that oocyte light subunits together with expressedrBAT or 4F2hc are responsible for the expression of sys- tine to cysteine should favor this direction of the ex-

change. The zwitterionic amino acids released to the tubu-tems bo,/-like and y/L-like, respectively. This, togetherwith the heterodimeic structure demonstrated for 4F2hc lar lumen should be reabsorbed via active transporters

(e.g., the sodium-dependent system neutral brush border)or indirectly evidenced for rBAT, suggests that the func-


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located in the apical plasma membrane of tubular epithe- sequences specifically reduces this amino acid transportactivity in OK cells (374). System bo,/-like activity has alsolial cells. Is this model valid? The fact that mutations in

the rBAT gene cause cystinuria, aminoaciduria of cystine been described in brush-border membrane vesicles fromchicken jejunum (560) and in Caco-2 cells (557). The spe-and dibasic amino acids, but not of zwitterionic amino

acids, clearly favors this model. cific expression of rBAT in the microvilli of the S3 seg-ment of the nephron and the cystinuria-specific mutationsIn addition to the cystine and dibasic amino acid re-

absorption defect of classic cystinuria, the present knowl- found in the rBAT gene allows us to propose that systembo,/-like (associated with rBAT) participates in the renaledge of cystine reabsorption in kidney and intestine is

very confused (for review, see Refs. 487 and 496). Work and intestinal reabsorption of cystine and dibasic aminoacids of high affinity, most probably with a tertiary activewith brush-border membrane preparations from rat kid-

neys showed that L-cystine reabsorption is less sodium transport mechanism (see above). Because of the locationof rBAT in the S3 segment of the nephron, where only adependent than that of other zwitterionic amino acids

(153, 352, 353, 449). Because of the weak sodium depen- part of the cystine reabsorption occurs (496), system bo,/-like could be envisaged as a low-capacity high-affinity sys-dence of L-cystine reabsorption, cystine and dibasic amino

acids may be accumulated across the apical membrane of tem of physiological relevance as revealed by its alterationin cystinuria. In conclusion, most probably rBAT/systemkidney epithelial cells partly because of the intracellular

reduction of cystine to cysteine and the negative mem- bo,/-like corresponds to the high-affinity reabsorption sys-tem of cystine described in renal (‘‘pars recta’’) and intesti-brane potential, respectively; basolateral transport sys-

tems would mediate the efflux of these amino acids (496). nal preparations (see above). In contrast, the proteinsresponsible for the high-capacity low-affinity reabsorptionSegal and co-workers (352, 486) have provided evidence

that renal brush-border membrane vesicles show two cys- of cystine in the proximal convoluted tubule are unknown.We are far from establishing the physiological role oftine transport systems: one with high-affinity (Km in the

micromolar range), shared with dibasic amino acids that 4F2hc. It is even possible to imagine a multifunctionalrole for this protein. Before the linkage between 4F2hcshows heteroexchange diffusion, and the other with low

affinity and unshared. In addition, several authors have and amino acid transport, it was implicated in calciummovement through the plasma membrane: 1) an anti-found inhibition by zwitterionic amino acids of cystine

uptake, measured at low concentration (micromolar 4F2hc monoclonal antibody (44D7) inhibited sodium/cal-cium exchanger activity in cardiac and skeletal musclerange) in renal brush-border preparations or perfused tu-

bules, suggesting that the high-affinity system is also sarcolemmal vesicles (for review, see Ref. 303). 2) Ananti-4F2hc antibody on parathyroid cells produces an in-shared by zwitterionic amino acids (153, 158, 449, 479). In

contrast to renal preparations, cystine transport in brush crease in cytosolic free calcium concentration at low ex-tracellular calcium levels (427). Recently, 4F2hc has alsoborder from mucosa of the small intestine shows a single

kinetic transport system of high affinity, shared with diba- been implicated in cell fusion (396) and regulation of cellsurvival/death control (592). Fusion regulatory proteinsic amino acids (404). Therefore, this high-affinity system,

present in kidney and intestine, may be the system that is (FRP-1) regulates virus-mediated cell fusion and fusion ofmonocytes. Purification and partial sequencing of humandefective in cystinuria (111, 555). Microperfusion studies

showed that this cystine high-affinity transport system is FRP-1 revealed a strong homology of the NH2 terminuswith human 4F2hc (it corresponds to the cluster of differ-present in the proximal straight tubule (S3 segment),

whereas the low-affinity system is present in the proximal entiation CD98) (11 of 15 amino acid residues are identi-cal); both proteins show cross-reactivity with differentconvoluted tubule (S1-S2 segments) (479). Recently, Ri-

ahi-Esfahani et al. (449) reported that luminal membrane antibodies, and the expression of both proteins is inducedby concanavalin A or interleukin-2 treatment (396). To us,vesicles from the pars recta (‘‘outer medulla’’) of rabbit

kidney show a conspicuous component of cystine trans- it seems that FRP-1 and 4F2hc are highly homologous,although not identical. To our knowledge, more extendedport of high affinity (Km values of Ç30 mM); interestingly,

cystine transport in the pars recta is less sodium depen- sequences of FRP-1 have not been reported. In addition,treatment of monocytes with anti-4F2hc antibodies re-dent and more sensitive to inhibition by micromolar con-

centrations of zwitterionic amino acids than in the pars sulted in cell fusion and formation of multinucleated giantcells of Cd/U2ME-7, a CD4/U97 cell line transfected withconvoluta (i.e., in apical membranes isolated from the

‘‘outer cortex’’). HIVgp160 gene, whereas other anti-4F2hc antibodies sup-press these induced fusion events (396, 397). Similarly,More recently, it was demonstrated that cystine is

transported through the apical pole of the ‘‘renal proximal anti-FRP-1/4F2hc antibodies suppress human parainflu-enza virus type 2-induced cell fusion (398). In a searchtubular’’ cell line OK via system bo,/-like (i.e., sodium-

independent, high-affinity transport system, shared with for cell surface markers expressed on hematopoietic stemcells, Palacios and co-workers (592) found that Joro 177dibasic and zwitterionic amino acids), and it is due to

the expression of rBAT; expression of antisense rBAT monoclonal antibody stained these cells. A cDNA library


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search with this antibody resulted in the cloning of mouse activity. Unfortunately, all our attempts to coexpress sys-tem y/ transport activity by coinjecting human 4F2hc and4F2hc. Interestingly, this antibody stimulates tyrosine

phosphorylation of an unidentified 125-kDa protein, in- mouse CAT-1 or CAT2-a failed (142). Therefore, at pres-ent, we do not known which specific cationic transportduces homotypic aggregation of progenitor lymphoid

cells, inhibits cell survival/growth of hematopoietic cells, activity is physiologically related to 4F2hc [i.e., systemssimilar to y/L (42, 146, 599), L (69), or y/ (88)]. In anyinduces apoptosis, and prevents the generation of

lymphoid, myeloid, and erythroid lineage cells. This study case, the obligatory exchange of amino acids via systemy/L-like, associated with 4F2hc expression in oocytes,suggests that 4F2hc might act as a membrane receptor

involved in the control of cell survival/death of hematopoi- which has been discussed in the previous sections, mighthave important physiological consequences. It has beenetic cells (592).

The above-mentioned roles of 4F2hc in cell fusion reported that efflux across the basolateral membrane isthe rate-limiting step in the intestinal absorption of dibasicand aggregation might involve integrin function. Very re-

cently, 4F2hc has been implicated in the regulation of amino acids (86, 383). Furthermore, leucine at low micro-molar concentration increases (6- to 10-fold) the transepi-integrin function (146): 1) expression of 4F2hc (i.e., CD98)

complements dominant suppression due to the overex- thelial flux of lysine (86, 87). Countertransport betweenlysine (outward) and leucine (inward) or allosterism waspression of an integrin b1-cytoplasmic domain, 2) 4F2hc

coimmunopreipitates with active b1-integrins, and 3) anti- considered to be responsible for this process. System y/Lcan sustain lysine-leucine exchange with an apparent Kmbody-mediated cross-linking of 4F2hc stimulated b1-inte-

grin-dependent cell adhesion. In this sense, anti-b2- and for leucine of Ç10 mM in the presence of sodium (127).If such a system is found in the basolateral membranesanti-b1-integrin antibodies blocked anti-FRP/4F2hc anti-

body-induced cell aggregation and antibody-induced poly- of intestinal or renal epithelial cells, the hypothesis thatsystem y/L can affect countertransport will be supportedkaryocyte formation, respectively (530). In any case, this

issue is not yet clear because other proteins also associate (14). The surface antigen 4F2hc has a basolateral localiza-tion in renal epithelial cells from the proximal tubulewith 4F2hc. Thus FRP-1/4F2hc and cytoskeletal proteins

(e.g., actomyosin, vimentin, and heat shock cognate pro- (436). System y/L-like, associated with 4F2hc expression,could be responsible for the active release of dibasictein 70) are coimmunoprecipitated by anti-FRP-1/4F2hc

antibodies (522), and anti-FRP-1/4F2hc antibodies change amino acids through the basolateral membrane of epithe-lial cells. The fact that the direction of exchange thatthe immunofluorescence pattern of these cytoskeletal

proteins (522). It is therefore difficult at present to ascer- is favored is L-arginine (outward) with low micromolarconcentration of leucine (inward) in the presence of so-tain whether anti-FRP-1/4F2hc antibody-mediated cell fu-

sion events are due to direct or indirect effects via dium strongly supports this hypothesis (90). Further re-search is needed to elucidate the mechanism (e.g., a weakchanges in the cell surface distribution or conformation

of other proteins. The role of the interaction of 4F2hc (or interaction of zwitterionic amino acids from inside due tothe low intracellular concentration of sodium) responsi-FRP-1) with other proteins (e.g., cystoskeletal proteins)

in any of the putative functions of 4F2hc is also unknown. ble for this asymmetric exchange.As mentioned before, several labs have observed in-

duced amino acid transport activity in oocytes injectedIII. INHERITED DISEASES OF PLASMAwith 4F2hc. One important question to resolve is the

MEMBRANE AMINO ACID TRANSPORTamino acid transport activity associated physiologicallywith 4F2hc in the cells that express 4F2hc naturally. Inthis sense, 4F2hc was originally described as a marker for This section deals with the inherited pathology due

to defective amino acid transport in the plasma membranetumor cells and activated lymphocytes (204). Stimulatedlymphocytes (e.g., by concanavalin A, interleukin-2, or of human cells. Table 10 summarizes the characteristics

of the defective amino acid transport systems and thephytohemagglutinin) have a larger increment (Ç60-fold)of 4F2hc in the plasma membrane (for review, see Ref. candidate genes for eight (including subtypes) of these

diseases. They are all aminoacidurias and therefore affect303); in some instances, this is due to increased transcriptstability (554). Boyd and co-workers (88) addressed this the tubular reabsorption of specific amino acids. For back-

ground information (clinical, genetic, biochemistry, andquestion (88): 1) phytohemagglutinin induces in lympho-cytes cationic amino acid transport with system y/ char- physiology) about these diseases, see Reference 476 and

OMIM (On-line Mendelian Inheritance in Men; http://acteristics; and 2) transfection of antisense oligonucleo-tide sequences of human 4F2hc and CAT-1 (system y/; www3.ncbi.nlm.nih.gov/omim/). Only one human amino

acid transporter gene, rBAT (also named SLC3A1, for sol-see sect. IIA), singly or in combination, inhibits the phyto-hemagglutinin-induced system y/ activity in human pe- ute carrier family 3, member 1; OMIM no. 104614), has

been shown to be responsible for one of these inheritedripheral blood mononuclear cells. These results suggesta shared responsibility of 4F2hc and CAT-1 in system y/ diseases, cystinuria type I (see below). Very recently (49,


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TABLE 10. Inherited diseases of plasma membrane amino acid transport

Defective Amino Acid Transport System Candidate Gene

Characteristics of Chromosome ChromosomeDisease Tissue affected transport locus Name Amino acid transport system locus Mutations

CystinuriaClassic

Type I Kidney and For CssC and aa/ 2p16.3-21 rBAT Exchanger bo,/-like 2p16-3-21 25 mutationsintestine (luminal) (high in humans

affinity)Type II Kidney and For CssC and aa/ 19q13.1 (?) For CssC and aa/ (?)

intestine (luminal)Type III Kidney For CssC and aa/ 19q13.1 For CssC and aa/ (low

(intestine?) (luminal) affinity) (?)Isolated Kidney For CssC (?) For CssC (?)

(intestine?)LPI Kidney, intestine Efflux for aa/ 14q For aa/ (?)

fibroblasts, (antiluminal)hepatocytes

Hartnup disorder Kidney and Na/ dependent ATBo (?) Na/ dependent for aao 19q13.3intestine for aao (luminal?)

(luminal)hph2 (?) Na/ dependent for aao (?) 11q13 hph2 mice

Iminoglycinuria Kidney Gly, Pro, OH-pro(high affinity)(?)

Dicarboxylic Kidney For aa0 (luminal) EAAT3 (?) Na/ and K/ dependent for 9p24 KO miceaminoaciduria (intestine?) aa0 (luminal?)

KO mice refers to null EAAT3 knockout reported by Stoffel and co-workers (416). CssC, cystine; aa/, dibasic amino acids; aao, zwitterionicamino acids; aa0, dicarboxylic amino acids. LPI, lysinuric protein intolerance. Question marks indicate ascription not well established; dashesindicate almost complete lack of information.

296, 595), cystinuria type III (perhaps also type II) and city participates in the selective motor neuron degenera-tion of the disease (457). Amyotrophic lateral sclerosis isLPI have been linked to chromosomes 19q13.1 and 14q,

respectively. The tissue distribution and the transport characterized by increased cerebrospinal fluid concentra-tion of L-glutamate and L-aspartate and a marked and spe-characteristics associated with the expression of the

sodium- and potassium-dependent zwitterionic amino cific decrease (Ç70%) in the Vmax of high-affinity glutamateuptake in synaptosomes from motor cortex and spinalacid transporter ATBo and the sodium- and potassium-

dependent anionic amino acid transporter EAAT3 (see cord (462). In this sense, decreased D-aspartate bindingsites have been reported in the spinal cord of ALS speci-sect. II) make them good candidates for Hartnup disorder

and dicarboxylic aminoaciduria, respectively (see Table mens (492), suggesting a decreased number of glutamatetransporters. Rothstein et al. (463) showed that this defect10). In addition, the dicarboxylic aminoaciduria devel-

oped by the null knockout EAAT3 mice (416) reinforces is specific to the glial EAAT2 transporter, the expressionof which is reduced up to 95% in the motor cortex andthe putative role of EAAT3 in this inherited disease. For

the rest of aminoacidurias due to defective renal reabsorp- spinal cord of postmortem samples from ALS patients.Despite the large loss of EAAT2 protein in those braintion (i.e., isolated cystinuria, hyperdibasic aminoaciduria

1, isolated lyinuria, and iminoglycinuria), neither obvious structures, the transcript levels of EAAT2 in motor cortexare not altered in ALS (66). The first mutational analysiscandidate genes nor chromosomal location is known.study showed no mutations in the EAAT2 mRNA sequenceIn addition to the above-mentioned inherited dis-

eases, it is worth mentioning the putative responsibility of ALS patients (347). In contrast, Rothstein found aber-rant EAAT2 RNA, including exon-skipping and intron-re-of the EAAT2 glutamate transporter in the sporadic form

of ALS. Amyotrophic lateral sclerosis is a chronic degener- tension species, in 65% of sporadic ALS specimens (J. D.Rothstein, personal communication). An intron-retentionative neurologic disorder characterized by the death of

motor neurons in the cerebral cortex and spinal cord. species has a dominant negative effect on the stability ofwild-type EAAT2 protein when expressed in COS cells.About 90% of ALS is sporadic, and only 10% is familial;

mutations in the superoxide dismutase-1 gene have been At present, the origin of these aberrant RNA species isunknown, and more likely, they are not genomic butfound in 15–20% of all familial cases (see entry no. 105400

in OMIM). Although the etiopathology of sporadic ALS is rather due to aberrant RNA processing. Further researchis needed to understand the mechanism underlying thenot known, it is hypothesized that glutamate excitotoxi-


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aberrant EAAT2 RNA processing in the brain of ALS pa- Refs. 408, 487). As described in section II, the transportcharacteristics of rBAT/bo,/-like system and its tissue andtients.subcellular distribution suggested the participation ofrBAT in a high-affinity reabsorption system of cystine and1. Cystinuriadibasic amino acids in kidney and intestine and postulatedrBAT as a good candidate gene for cystinuria. MutationalClassic cystinuria (OMIM no. 220100) is an inherited

hyperaminoaciduria of cystine and dibasic amino acids analysis of the rBAT gene of patients with cystinuria ini-tially revealed six missense cystinuria-specific mutations;(for review, see Refs. 351, 487), discovered by Wollaston

(604), and described as one of the first four ‘‘inborn errors for one of these mutations (Met467Thr; see Fig. 8), defec-tive amino acid transport activity was shown (see sect. II);of metabolism’’ by Garrod (163). Cystinuria is an autoso-

mal recessive disease with an overall estimated preva- this demonstrated that mutations in rBAT cause cystinuriaand that rBAT/system bo,/-like participates in the renallence of 1 in 7,000 neonates; prevalence estimations range

between 1 in 2,500 neonates in Israeli Jews of Libyan reabsorption of cystine and dibasic amino acids (78). Ge-netic analysis demonstrated linkage of cystinuria withorigin and 1 in 100,000 in Sweden (487). In our opinion,

these numbers are overestimations in screening pro- chromosome 2p microsatellite markers (430), which colo-calize with the rBAT gene locus in 2p16.3 (79). Furthergrams, because of the nonsilent hyperaminoaciduria phe-

notype of cystinuria types II and III (see below). Because mutational analysis by several groups (for review, seeRefs. 170, 408, 409) of the rBAT gene in Italian, Spanish,of the poor solubility of cystine, it precipitates to form

kidney calculi that produce obstruction, infection, and Middle Eastern, Eastern European, Canadian, Japanese,and United States populations revealed a growing numberultimately renal insufficiency. Three types of classic cysti-

nuria have been described (456): type I heterozygotes of cystinuria-specific mutations in the rBAT gene (25 mu-tations have been described, including missense, non-present normal aminoaciduria, whereas types II and III

present high and moderate hyperaminoaciduria of cystine, sense, splice-junction, deletions, and insertions; see Fig.8). Four cystinuria-specific rBAT mutants have beenlysine, and, to a lesser extent, arginine and ornithine. As a

consequence of the intestinal amino acid transport defect, shown to express defective amino acid transport activity(see sect. IID4 and Fig. 8; Refs. 78, 91, 366). Mutationstype I and II homozygotes do not show increase in the

plasma levels of cystine after an oral administration of Met467Thr and R270X [stop codon at arginine residue 270;this eliminates two-thirds of the protein toward the COOHthe amino acid. In contrast, type III homozygotes show a

nearly normal increase in the plasmatic levels of cystine terminus; Miyamoto et al. (367) reported that deletionsaffecting the COOH terminus result in defective rBAT-after the oral dose. This suggests that the amino acid

transport system affected in cystinuria type III is not ex- expressed amino acid transport activity] represent ap-proximately one-half of the cystinuric chromosomespressed or is not very conspicuous in the intestine. Others

(197, 377) divide cystinuria into two types: type I, or true where mutations have been detected. These mutationshave been found in homozygosis in several patients andrecessive, and type II, or incomplete recessive (this in-

cludes the types II and III of Rosenberg; Ref. 456). in compound heterozygotes with other mutations.Clinical and physiological evidence suggested hetero-Dent and Rose (124) postulated that cystinuria may

result from the defective function of a common uptake geneity in cystinuria (see above). 1) The oral cystine testmay be indicative that in type III cystinuria the intestinalsystem for cystine and dibasic amino acids. Milne et al.

(361) demonstrated a reduced intestinal absorption of di- defect is not very conspicuous. 2) Most of renal reabsorp-tion of cystine occurs in segments S1-S2 of the nephronbasic amino acids in patients with cystinuria. Finally,

transport studies in vitro demonstrated a defective accu- (i.e., in a tubular region other than that in which rBAT isexpressed). Thus other cystine reabsorption system(s)mulation of cystine and dibasic amino acids in biopsies of

patients with cystinuria (111, 555). Interestingly, patients not present (or not very conspicuous) in the small intes-tine may also be cystinuria genes (see sect. IID5). Muta-with cystinuria show no malabsorption of arginine when

given in a peptide form; this suggested normal apical ab- tional analysis suggested that only patients with type Icystinuria carried mutations in the rBAT gene (164, 217).sorption of peptides in cystinuria and positioned the dis-

ease-associated transport defect at the apical membrane Genetic linkage analysis with markers of the genomic re-gion of rBAT in chromosome 2 and intragenic markers ofof the intestinal epithelium (21). As discussed in section

IID5, there is an apical high-affinity amino acid transport rBAT have demonstrated genetic heterogeneity for cysti-nuria (80). The rBAT gene is linked to type I cystinuria,system for cystine and dibasic amino acids that also

shows interaction (cis-inhibition and heteroexchange) but not to type III (OMIM no. 600918). A wide searchthrough the genome, carried out independently by twowith zwitterionic amino acids, in the brush-border mem-

branes of the epithelial cells of the proximal straight tu- groups, localized type III (and perhaps also type II) cysti-nuria gene in patients from Italy and Israeli Jews with abules of the nephron and of the small intestine. It is be-

lieved that this is defective in cystinuria (for review, see Lybian origin to 19q13.1 (49, 595). We are currently analyz-


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ing this locus for the identification of a new cystinuria knowledge, linkage and/or mutational analysis of therBAT gene and the 19q13.1 cystinuria locus in isolatedgene. In these studies, the phenotype classification of cys-

tinuria (types I, II, and III) was based on the urine excre- cystinuria has not been reported.tion values of cystine and dibasic amino acids in the obli-gate heterozygotes. 2. Other dibasic aminoacidurias

Cystinuria type I, the most frequent worldwide (i.e.,ú60% of the cases), is due to mutations in the rBAT gene. There are four diseases in which a cationic amino

acid transport defect is suspected (for a review, see Ref.As discussed in section IID, this gene codes for a proteinthat most probably participates as a subunit of a hetero- 497): 1) cystinuria (see above); 2) LPI, hyperdibasic ami-

noaciduria type 2, or familial protein intolerance (OMIMdimeric bo,/-like transporter. This activity is responsiblefor the high-affinity cystine and dibasic reabsorption in no. 222700); 3) hyperdibasic aminoaciduria type 1; and

4) isolated lysinuria. Lysinuric protein intolerance is anthe S3 segment of the nephron and in the small intestinewith a tertiary active transport mechanism coupled with autosomal recessive trait. Almost one-half of the known

LPI patients (Ç100) are from Finland, where the preva-the exchange of neutral amino acids. Interestingly, in theItalian and Spanish patients with cystinuria type I sub- lence of the disease is 1 in Ç60,000. In contrast, hyperdi-

basic aminoaciduria type 1 (autosomal dominant trait)jected to study, we have identified mutants only in Ç50%of the cases. In the future, this figure may increase through and isolated lysinuria have been described only in one

French Canadian pedigree and in a Japanese patient, re-the analysis of deletions in the rBAT gene (we are cur-rently studying 3 putative new deletions), but it is possible spectively. Lysinuric protein intolerance was first de-

scribed by Perheentupa and Visakorpi (418). In additionthat mutations in the rBAT gene will not explain all cysti-nuria type I chromosomes. In this sense, the putative light to hyperdibasic aminoaciduria, the clinical symptoms of

LPI are failure to thrive, protein aversion, short stature,subunit of rBAT could also be envisaged as a type I cysti-nuria gene. From the cystinuria loading test (see above), hepatomegaly, osteoporosis, hyperammonemia, common

interstitial lung disease, and renal damage, and occasion-we can speculate that the type III cystinuria gene wouldhave a low expression in the small intestine. The transport ally moderate mental retardation. It is believed that the

disease is caused by a defective dibasic amino acid trans-system responsible for the high-capacity low-affinity reab-sorption of cystine in segments S1-S2 of the nephron may port that is expressed at the basolateral membrane of

the renal and intestinal epithelia, and in nonepithelial cellbe defective in cystinuria type III. In contrast, there is noobvious candidate gene for type II cystinuria. This is a types (e.g., culture fibroblasts, hepatocytes) (for review,

see Ref. 497). An oral loading administered to LPI patientsrare (°5% of the cystinuria cases) type of the disease,and its ascription to the 19q13.1 locus is at present not with the dipeptide lysyl-glycine increased plasma glycine

concentrations properly, but plasma lysine remained al-definitive, since this linkage, although significant, is basedon a small number of cases (49) (see Table 10). most unchanged; this indicated unaffected apical peptide

absorption and cellular hydrolysis and suggested the baso-Whether mutations in rBAT (chromosome 2p16.3)and in the new type III cystinuria locus (chromosome lateral location of the defective cationic amino acid trans-

port (443). In agreement with this, transport studies with19q13.1) lead to a full-blown type I/type III cystinuria phe-notype is still an open question. Initial mutational analysis jejunal LPI biopsy samples showed that the transport de-

fect is situated at the basolateral plasma membrane (125).suggested this genotypic/phenotypic interaction (164,217). Linkage analysis with both cystinuria loci is cur- In an interesting study, Scriver, Simell, and co-work-

ers (501) reproduced in LPI fibroblast cell lines the cat-rently in progress. Preliminary data suggest that cases oftype III heterozygotes within the lower range of cystine ionic amino acid transport defect; LPI fibroblasts showed

a reduced trans-stimulated efflux of cationic amino acids.and dibasic hyperexcretion values of these carriers (173)may be due to mutations in the rBAT gene. This defect showed gene-dosage effect (homozygotes

more affected than heterozygotes). It is believed that theFinally, Brodhel et al. (67) reported isolated cystinu-ria (OMIM no. 238200) in two siblings of unrelated parents defective cationic amino acid transport activity corre-

sponds to system y/, but unfortunately, this has not been(see Table 10), in which urinary hyperexcretion of aminoacids was restricted to cystine. This suggested that a cys- carefully characterized. Cationic amino acids are trans-

ported through the plasma membrane of human fibro-tine renal transporter not shared with dibasic amino acidswas defective in these patients (for review, see Ref. 487). blasts via systems y/ and y/L (see sect. I) (Torrents and

Palacın, unpublished data).Biochemical evidence for this transporter has not beenobtained in renal or intestinal transport studies (496). It Very recently, Simell, Aula, and co-workers (296) re-

ported a locus on chromosome 14 for LPI in Finnish pa-is therefore possible that a rare allele either of the rBATgene or of the cystinuria gene in 19q13.1 may be responsi- tients; linkage disequilibrium in markers within this locus

suggests that LPI in these patients is due to one historicalble for this phenotype (i.e., a mutant affecting cystinuriatransport but not dibasic amino acid transport). To our mutation. The hyperdibasic aminoaciduria characteristic


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of LPI has fostered studies on the involvement of the tryptophan in the urine, plasma, and feces as well as re-duced transient plasma levels increase after an oral loadknown cationic amino acid transporter in this disease.

Unfortunately, none of the known proteins involved in of zwitterionic amino acids. Then, the disorder appears toinvolve a renal reabsorption defect, and in some patientscationic amino acid transport seems to be responsible for

LPI. Indeed, human CAT-1 (chromosome 13q12–14), CAT- intestinal malabsorption of zwitterionic amino acids (forreview, see Ref. 304). Studies with brush border of intesti-2 (chromosome 8p21.3–22), and CAT-4 (chromosome

22q11.2) (see Table 1) have been excluded from linkage nal mucosa biopsies, but not with leukocytes or fibro-blasts, from Hartnup patients showed defective zwitter-to the LPI phenotype in Finnish patients (296). Similarly,

mutational and linkage analysis excluded human CAT-1, ionic amino acid transport (178, 493, 532, 545).Scriver and co-workers (474, 477) have proposed thatCAT-2, and CAT-4 as LPI genes among Italian or Japanese

LPI patients (128, 224, 485). The recently described rat Hartnup disorder is an amino acid transport single genedefect affecting kidney and intestine, with a variant formCAT-3 (219), for which no human counterpart has been

cloned (see Table 1), is expressed exclusively in brain affecting only the kidney; in contrast, the pathologicalstate associated with the disease (niacin deficiency)and therefore does not represent a candidate gene for

LPI. Two other proteins are known to be associated with seems to be multigenic. These authors suggest that othergenes that control plasma amino acid homeostasis maycationic amino acid transport: rBAT and 4F2hc (see sect.

IID). The rBAT gene is expressed in kidney and intestine, influence the occurrence of clinical abnormalities withthe Hartnup biochemical defect. The disease symptomsand it is associated with the cystinuria phenotype (see

above). In contrast, the putative role of 4F2hc in the renal occur with low aggregate plasma amino acid levels andnutritional stress (malnutrition, diarrhea).reabsorption and intestinal absorption (basolateral efflux

of cationic amino acids by exchange with zwitterionic Very recently, Dove and co-workers (528) developeda mouse model for Hartnup disease (hyperphenylalanin-amino acids plus sodium; see sect. IID5) makes it a good

candidate for LPI. In addition, 4F2hc is expressed in fi- emia 2; hph2) by N-ethyl-N-nitrosourea mutagenesis andscreening for delayed plasma clearance of an injected loadbroblasts (Torrents and Palacın, unpublished data), where

the LPI transport defect has been substantiated (501). of phenylalanine. The hph2 is a recessive mutation thatcauses a deficient amino acid transport that is similar but4F2hc does not seem to be directly involved in LPI, since,

as indicated above, LPI gene localizes to chromosome 14q not identical to Hartnup disease. Like Hartnup patients,the hph2 homozygotes show 1) specific urinary hyperex-(296), and the human 4F2hc gene localizes to chromo-

some 11q12–13 (174). However, a role of 4F2hc in LPI cretion of many of the zwitterionic amino acids, whileplasma concentrations of these amino acids are normal;cannot be ruled out. As mentioned in section IID, there is

evidence that the functional unit of 4F2hc/system y/L-like 2) a partial deficiency in the sodium-dependent uptake ofglutamine in brush-border membrane vesicles; and 3) atransporter is composed by 4F2hc (heavy chain) plus an

unidentified light subunit (142). This putative light subunit niacin-reversible syndrome influenced by diet and geneticbackground. In contrast to Hartnup patients, hph2 homo-might be envisaged as an LPI gene. The identification of

the LPI gene in the 14q locus (already restricted to 100 zygotic mice show urine hyperexcretion of arginine, amild urine hyperexcretion of tryptophan and valine, andkb) and/or the cloning of the putative light subunit of 4F2

surface antigen may clarify this issue in the near future. significant urine hyperexcretion of methionine.Dove and co-workers (527) mapped hph2 to a region

of mouse chromosome 7 synthenic with human chromo-3. Hartnup disordersome 11q13 (see Table 10). Interestingly, 4F2hc, the puta-tive subunit of the amino acid transport system y/L-like,This disorder (OMIM no. 234500) was first described

by Baron et al. (35). It is transmitted as an autosomal also maps to this locus (see Table 1, rBAT). This aminoacid transporter-related protein has been suggested as arecessive trait, and it is characterized by a pellagra-like

light-sensitive rash (niacin deficiency), cerebellar ataxia, candidate gene for the Hartnup disorder (304). In ouropinion, the amino acid transport associated with 4F2hcemotional instability, and aminoaciduria. This is a charac-

teristic aminoaciduria that involves the zwitterionic amino expression in oocytes (systems y/L-like or L-like, de-pending on the authors; see sect. IID5), and the almostacids (with the exception of cysteine/cystine, glycine, me-

thionine, and the imino acid proline) and that occurs at ubiquitous tissue distribution and the renal basolaterallocalization of 4F2hc (see sect. IID) weakens the candida-a frequency of 1 in Ç40,000 in urine amino acid screens

(for review, see Ref. 304). Most of the hyperexcretors ture of this gene for this disorder. In any case, becauseof the hitherto unclear physiological role of 4F2hc innever display the niacin deficiency symptoms, and there-

fore, the Hartnup disorder is usually benign (477, 300). amino acid transport (see sect. II) and the dissimilar ami-noaciduria phenotypes (i.e., urine excretion values of argi-Urinary hyperexcretion occurs with normal amino acid

plasma levels. Some patients have elevated fecal amino nine, tryptophan, valine, and methionine; see above) ofthe hph2 mice with Hartnup patients, it will be very infor-acid levels and secondary metabolites of the excess of


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mative to answer the following questions: 1) What renal the father and two sons had hyperglycinuria. The renaltubular titration curve for proline reabsorption in one ofamino acid transport activity is defective in the the hph2

mice? 2) Does the hph2 locus contain the mouse 4F2hc the sons was compatible with a mutation affecting theaffinity of the proline transporter. This ‘‘Km’’ variant hasgene? If the answers to these questions reinforce the can-

didature of 4F2hc for the hph2 phenotype, it should be been designated iminoglycinuria type II (OMIM no.138500). It is believed that all these variants are allelic:assessed directly.

The transport characteristics (sodium-dependent the same renal phenotype is observed in probands inher-iting two recessive mutant alleles, two hyperglycinuriczwitterionic amino acid transport) and the epithelial dis-

tribution of ATBo (see sect. IIC) fit those expected for the alleles, or two different alleles (475).There is evidence of two sodium-dependent prolinetransporter responsible for the Hartnup disorder (Table

10). In contrast to the human synthenic locus of the hph2 transport systems in the brush border of human renalcortex (154): a high-affinity system shared with glycinemouse mutation (chromosome 11q13), the ATBo gene lo-

calizes to 19q13.3 (see Table 6). Therefore, ATBo does not and a low-affinity system not shared with glycine. Studiesin rat, dog, and rabbit kidneys (reviewed in Ref. 89) re-seem to hold the hph2 mutation. In our opinion, because

of the nonidentical aminoaciduria phenotype of Hartnup vealed two sodium-dependent transport systems for iminoacids and glycine, one of high affinity and specific fordisorder and hph2 mutation, there is still room for a role

of the ATBo gene in the Hartnup disorder. First, direct these substrates, and the other with low affinity and withbroad specificity with other zwitterionic amino acids. Forevidence for the apical localization of the ATBo trans-

porter in renal and intestinal epithelia and for an active glycine, two apical sodium-dependent transport systemshave been described: a high-affinity low-capacity systemtransport mechanism for the amino acid transport activity

associated with ATBo should be offered. Then, direct ge- located in the proximal straight tubules and a low-affinityhigh-capacity system in the proximal convoluted tubule.netic analysis of the ATBo gene (mutational and/or linkage

studies) in Hartnup’s aminoaciduria families should be Notice the similarity with the proposed renal reabsorptionsystems for cystine (see sect. IID5). It is hypothesized thataddressed.the defective transport system is low-capacity high-affinityfor glycine and the two imino acids in the proximal4. Iminoglycinuriastraight tubule, but there is no direct proof of this (89).Ontogeny in humans also gives clues to the amino acidFamilial iminoglycinuria (OMIM no. 242600) is a be-

nign inherited defect of membrane transport (for review, transport systems serving the renal reabsorption of theseamino acids (reviewed in Ref. 89): 1) maturation of thesee Ref. 89). It involves a glycine, L-proline, and hydroxy-

L-proline transporter in the renal tubule and, in some renal reabsorption of glycine and proline occurs at differ-ent times after birth. 2) In contrast to controls, iminoglyci-cases, in the epithelial intestine. There are no reports of

the prevalence of this disease, but it seems more frequent nuria homozygotes have an almost complete absence oftubular reabsorption for proline and glycine; with matura-in Ashkenazim (see OMIM). As for other systems of renal

reabsorption of amino acids, the reabsorption of these tion of the tubular function, reabsorption of proline andglycine appear independently. 3) In rats, the postnatalamino acids matures during the first months of life. The

persistence of iminoglycinuria beyond 6 mo is considered prolinuria is associated with low activity of a high-affinitysodium-dependent nephron transport system. This and ad-abnormal. In addition to familial iminoglycinuria, this uri-

nary hyperexcretion phenotype also occurs in familial ditional evidence suggest that ontogeny is associated withdeficient activity of high-affinity systems for imino acidshyperprolinemia and hyperhydroxyprolinemia, and in the

generalized disturbance of membrane transport of the and glycine that does not include the system controlledby the familial iminoglycinuria gene (89).Fancony syndrome. In contrast to these, urine hyperex-

cretion of glycine, L-proline, and hydroxy-L-proline in fa- Unfortunately, there is no genetic information of achromosome locus for the familial iminoglycinuria pheno-milial iminoglycinuria is specific to these amino acids and

occurs with normal levels of these amino acids in plasma. type. The transport characteristics of the expected aminoacid transport system defective in this phenotype fit thatFor glycine and these imino acids, the endogenous renal

clearance rates are high, and the net reabsorption de- of the IMINO and Proline transport systems (see sect. I).Three cDNA and their splice variants, which belong tocreased in familial iminoglycinuria probands (reviewed in

Ref. 89). the superfamily of sodium- and chloride-dependent neuro-transmitter transporters, GLYT1, GLYT2, and PROT (seeThe iminoglycinuria phenotype is autosomal reces-

sive, but in some pedigrees, there is an incomplete reces- sect. II), transport glycine and/or proline with characteris-tics of these systems (see Table 5). At present, it seemssive phenotype; of 16 familial iminoglycinuria pedigrees

reviewed by Chesney (89), in 9 pedigrees the obligate that GLYT2 and PROT are specific to the CNS, and onlya peripheral tissue distribution has been demonstrated forheterozygotes show hyperglycinuria without prolinuria. In

addition, Greene et al. (177) reported a family in which GLYT1 (see Table 5). The splice variant 1a of GLYT1 is


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expressed in kidney and other peripheral tissues; in lung, Relevant amino acid transport systems, like systems A, L,spleen, and liver, there is evidence that GLYT1–1a is ex- N, and x0c , have not been identified at the molecular level.pressed in macrophages and not in the parenchymal cells We are just beginning to understand the molecular bases(62). To our knowledge, there are no data on the subcellu- of the human inherited diseases of amino acid transport:lar distribution of GLYT1–1a in kidney. If this transporter mutations in the rBAT gene cause cystinuria type I. Onis expressed in the apical pole of the tubular epithelium, it the other hand, the first knockouts for amino acid trans-would be a good candidate for the familial iminoglycinuria porters have been produced, by homologous recombina-phenotype. It is worth mentioning that L-proline uptake tion for the cationic amino acid transporter CAT-1 andin renal brush border is chloride dependent in addition to for the glutamate transporters EAAT2 and EAAT3, andsodium dependent (478). The human GLYT1 gene local- by antisense technology for the glutamate transportersizes to chromosome 1p31.3-p32 (see Table 4). Linkage EAAT1, EAAT2, and EAAT3 in brain and for the rBAT/studies of the familial iminoglycinuria phenotype would system bo,/-like in epithelial renal cells. We can envisagebe the first step to contrasting this hypothesis. a final goal in this line of research: the ascription of every

amino acid transporter and the cognate transport system5. Dicarboxylic aminoaciduria to the macroscopic fluxes of amino acids across the

plasma membrane of mammalian cells.Teijema et al. (552) reported the first case of dicar-The amino acid transporters cloned can be groupedboxylic aminoaciduria (OMIM no. 222730) in a female

in four protein families, and for many amino acid trans-child, most probably due to an anionic amino acid trans-port systems, several transporter isoforms have beenport defect in kidney and intestine. To our knowledge,identified. This has revealed a high complexity in mam-only two other cases have been reported (355, 526).malian amino acid transport. A relevant question, then,Swarna et al. (526) found one of these by screening foris what are the key structural elements that explainamino acid disorders in 500 mentally retarded children inamino acid transport mechanisms at the molecularIndia. Melancon et al. (355) detected in a neonatal screen-level? After the cloning of the first mammalian aminoing program a boy with massive glutamic and asparticacid transporters, a growing number of studies basedaminoaciduria. The boy was apparently healthy at the ageon site-directed mutagenesis and chimera constructionsof 3 years. Amino acid clearance studies revealed the pres-are being reported. These studies, although valuable,ence of renal wastage of dicarboxylic amino acids. Intesti-show a weakness, the lack of knowledge of the three-nal transport and in vitro oxidation of dicarboxylic aminodimensional structure of these transporters sitting in theacids were found to be intact. The same group later re-plasma membrane. There is no doubt that an enormousported (356) reduced uptake velocities of glutamate andchallenge in this line of research is the resolution of theaspartate in dicarboxylic aminoaciduria fibroblasts.amino acid transporter structures at the Ao scale, as forThe neuronal and peripheral high-affinity glutamate

transporter EAAT3 (see Tables 6 and 7) is an obvious aquaporin-1 (587).candidate for the transporter defective in dicarboxylic Finally, two amino acid transport systems, bo,/-likeaminoaciduria (see sect. IIC). This transporter is highly and probably y/L-like, could be a heterodimeric structureexpressed in kidney and in epithelial small intestinal cells composed of rBAT or 4F2hc, respectively, plus the corre-(19, 245). Indeed, EAAT3 is the only known anionic trans- sponding as yet unidentified subunit. If this hypothesis isporter in kidney and intestine (see Table 7). Finally, Stof- proven, these transporters will be the first known trans-fel and co-workers (416, 517) reported that null knockout porters for organic solutes with a heteroligomeric struc-EAAT3 mice develop dicarboxylic aminoaciduria. This ture.clearly substantiates the role of EAAT3 transporter in the In the present decade, molecular biology has reachedrenal reabsorption of anionic amino acids and in addition mammalian amino acid transport; now we are on the waysuggests EAAT3 gene (chromosome 9p24; see Table 6) as to explaining interorgan amino acid flux at the molecularthe immediate candidate for dicarboxylic aminoaciduria level.(Table 10). At this stage, because of the low number ofdisease cases described, the obvious next step is to search We thank Drs. Carol MacLeod, Baruch Kanner, Enerstfor mutations of the EAAT3 gene in patients with dicar- Wright, Pertti Aula, Cecilio Gimenez, Rosa Deves, Marcal Pastor-

Anglada, Eduardo Soriano, Josep L. Gelpı, Virginia Nunes, Bea-boxylic aminoaciduria.triz Lopez-Corcuera, and David Torrents for helpful discussionfor the writing of this manuscript. Our most grateful thanks to

IV. PROSPECTS Dr. Gianfranco Sebastio for access to nonpublished informationon the putative human CAT-4 transporter, to Carles Pucharcos

We are halfway toward the identification of the genes for help in consulting data bases via internet, and to Ceciliocoding for the transporters that mediate the amino acid Gimenez for access, before publication, to the manuscript for

Reference 622. We also thank all our collaborators from Spain,flux across the plasma membrane of mammalian cells.


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