Proc. Natl. Acad. Sci. USAVol. 91, pp. 247-251, January 1994Cell Biology

The lung amiloride-sensitive Na+ channel: Biophysical properties,pharmacology, ontogenesis, and molecular cloning

(human sequence/cystic flbrosis)

NICOLAS VOILLEY*, ERIC LINGUEGLIA*, GuY CHAMPIGNY*, MARIE-GENEVIEVE MATTtIt,RAINER WALDMANN*, MICHEL LAZDUNSKI*t, AND PASCAL BARBRY**Institut de Pharmacologie Moleculaire et Cellulaire, Centre National de la Recherche Scientifique, Universit6 de Nice Sophia Antipolis, 660 route desLucioles, Sophia Antipolis, F-06560 Valbonne, France; and tCentre de Genetique Medicale, Physiopathologie Chromosomique, Hopital d'Enfants de laTimone, Boulevard Jean Moulin, 13385 Marseille cedex 05, France

Communicated by Pierre Joliot, September 20, 1993

ABSTRACT Water balance in the lung is controlled viaactive Na+ and Cl- transport. Electrophysiological measure-ments on lung epithelial cells demonstrated the presence of aNa+ channel that is inhibited by amiloride (Ko.5 = 90 nM) andsome of its derivatives such as phenamil (Ko.5 = 19 nM) andbenzamil (Ko.5 = 14 nM) but not by ethylisopropylamiloride.An amiloride-sensitive Na+ channel of 4 pS was recorded fromoutside-out patches excised from the apical membrane. Thischannel is highly selective for Na+ (PNa+/PK+ 2 10). Isolationof a human lung cDNA led to the primary structure of the lungNa+ channel. The corresponding protein is 669 residues longand has two large hydrophobic domains. An amiloride-sensitive Na+-selective current apparently identical to the oneobserved in lung epithelial cells was recorded after expressionof the cloned channel in oocytes. The level of the mRNA for theNa+ channel was highly increased from fetal to newborn andadult stages. This observation indicates that the increased Na+reabsorption that occurs at birth as a necessary event to passto an air-breathing environment is probably associated withcontrol of transcription of this Na+ channel. The human genefor the lung Na+ channel was mapped on chromosome 12pl3.

During fetal life, the lungs are filled with liquid, which arisesfrom continuous secretion of epithelial cells (1, 2). At birth,the pulmonary epithelium changes its predominantly activeCl--secreting properties for predominantly active Na+-absorbing properties, the result being the clearance of thepulmonary fluid as the lung switches to an air-conductingsystem (1, 2). In adults, Na+ transport participates in controlof the quantity and composition of the respiratory tract fluidthat is necessary for gas exchange and prevention of alveolarcollapsing and particle deposition. A number of data suggestthat Na+ absorption by alveolar epithelium mainly occursthrough amiloride-sensitive pathways, probably channels,located at the apical membrane oftype II pneumocytes (3-6).Surprisingly, available electrophysiological measurementswith the patch-clamp technique (7) as well as studies of22Na+flux into pneumocyte vesicles (8) or binding studies withlabeled derivatives of amiloride such as [3H]benzamil (9) or[3H]bromobenzamil (10), which are known to be specificblockers of epithelial Na+ channels (NaCh) (11), suggest thatthe amiloride-sensitive NaCh in pneumocytes would be quitedifferent from other NaCh identified in the renal collectingtubule and/or in the renal pars recta (refs. 12 and 13;reviewed in ref. 11).Because of the importance of the NaCh for pulmonary

function, because abnormalities of channel function havebeen identified in cystic fibrosis (14), and because amilorideis presently assayed as a potential treatment for this major

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

genetic disease (15), it appeared important (i) to analyze indetail the biophysical and pharmacological properties of theamiloride-blockable channel in type II pneumocytes, (ii) todetermine the structure of the human channel by molecularcloning and identify the chromosomal localization of theNaCh gene, and (iii) to study its transcription during devel-opment.§

MATERIALS AND METHODSCell Culture. Primary cultures of fetal alveolar type II cells

were carried out according to Orser et al. (16). The cells wereseeded in minimal essential medium with D-valine, 0.1 mMnonessential amino acids (Sigma), and 10% fetal calf serum(Boehringer Mannheim) and studied 24 hr to 8 days afterplating.

Electrophysiological Experiments. Resting membrane po-tential and Na+ current were recorded at room temperaturewith the whole-cell patch-clamp technique (17). Outwardcurrent refers to the flow of cations from the pipette (cyto-solic side) to the bath (external side). The pipette solutioncontained 100 mM potassium gluconate, 40 mM KCl, 2 mMMgCl2, 0.5mM CaCl2, 4 mM EGTA/KOH (10nM free Ca2+),2 mM K2ATP, and 10 mM Hepes (pH 7.2). The bath solutioncontained 140 mM NaCl, 1 mM MgCl2, 1.8 mM CaCl2, and 10mM Hepes (pH 7.2). The low Na+ solutions were obtained bysubstituting NaCl in the bath solution with equal amounts ofcholine chloride.

Single-channel currents were recorded from outside-outmembrane patches (17). In most experiments, solutions weresimilar to those used in the whole-cell configuration. Forexperiments in Na+ symmetrical conditions, K+ in the pi-pette solution (see above) was replaced by Na+. Single-channel data were filtered at 200 Hz. The open probability(P.) was calculated according to ref. 18.cDNA Library Construction, Screening, and Sequencing. A

cDNA library was synthesized (19) from human adult lungpoly(A)+ RNA in LambdaZAP II phage (Stratagene). Clones(3 x 105) were plated, and five plaques hybridizing with a614-bp restriction fragment obtained from the rat colon NaCh(20) by digestion with EcoRI and Kpn I were identified andpurified.For sequencing, DNA was prepared by deletion with the

Erase-a-Base system from Promega and sequenced in bothdirections by dideoxynucleotide sequencing using the dye

Abbreviations: NaCh, Na+ channel(s); HLNaCh, human lung NaCh;LE, lung epithelial; EIPA, ethylisopropylamiloride; EPA, ethylprop-ylamiloride.1To whom reprint requests should be addressed.§The sequence reported in this paper has been deposited in theGenBank data base (accession no. X76180).

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terminator kit and an automatic sequencer (Applied Biosys-tems model 373A).Northern Blot Analysis. Northern blotting was carried out

by standard techniques (21). The membrane was hybridizedwith restriction fragments (an EcoRI/Kpn I 614-bp fragmentof the rat colon clone and a Sau I/HincII 814-bp fragment ofthe human lung clone) labeled with [a-32P]dCTP by randompriming.Human Gene Mapping by in Situ Hybridization. In situ

hybridization was carried out on chromosome preparationsobtained from phytohemagglutinin-stimulated human lym-phocytes cultured for 72 hr according to ref. 22.

Oocyte Preparation, Microni ection, and Electrophysiolog-ical Measurements. These procedures were carried out asdescribed (23). cRNA synthesis ofeach clone was performedwith a Stratagene kit. Capped cRNA (1 u.g/,ul) was injectedinto oocytes (50 nl per oocyte). The Na+ selectivity wastested in the usual buffer without Na+ (Na+ replaced by K+).

RESULTSElectrophysiological Studies of the NaCh in Rat Pulmonary

Cells. Patch-clamp experiments were performed on primarycultures of lung epithelial (LE) cells. Fig. lAa shows that

Aa b 14ONa 1/2Na l4ONa ONa l4ONa

20 2i min

1MU#-s

A)0

miOC AMILORDE d a OAMILRDE

0 A"LI . EIPAImin -0-

lqsM -10-9 -8 -7 -6 -5 -4 -3B

a b log(concentration)

OpA-s..d

OpA- ___2__ __A___a ~~AF 14ONa MNa ONa b4ONA

o AMILORIDEE 10 rOPPEAI. A BENZAML/rL. V EIPAW ff

t -lIT9 -8 -7 -6 -s -4 - 3log(concentration)

C 1ONM

0pA-m.111

IOOpArAMLORIDE

FIG. 1. Electrophysiological analysis of the amiloride-sensitiveNaCh in cultures of alveolar type II pneumocytes. (A) (a) Restingmembrane potential (Er) recorded on monolayer in the whole-cellconfiguration. Membrane resistance was measured by current pulsesof -20 pA every 5 s. (b) Membrane potential recording at differentexternal Na+ concentrations with the effects of amiloride. (c) Effectof increasing doses of amiloride on the resting potential. (d) Dose-response curves for phenamil, amiloride, and ethylisopropyl-amiloride (EIPA). (B) (a) Effect of amiloride (10 uM) on the currentrecorded at -20 mV. (b) Dependence of the amiloride-induceddecrease of current recorded at -20 mV on the external Na+concentration. (c) Effect of increasing doses of amiloride on thecurrent recorded at -20 mV; downward deflections correspond toinward current produced by single pulses from -20 to -60 mV andof 400-ms duration. (d) Dose-response curves for phenamil, ben-zamil, amiloride, and EIPA measured on the current recorded at -20mV. Horizontal bars indicate addition of amiloride (Ami).

addition of amiloride to cells in a 140 mM Na+ mediuminduced a reversible hyperpolarization. No change in mem-brane conductance could be observed, suggesting a cell-to-cell electrical coupling. Indeed, in the presence of differentblockers of gap junctions (such as 1-heptanol), amilorideproduced an increase of cell resistance (data not shown). Themean resting potential was -6.5 ± 13.8 mV (mean ± SE; n= 90) and amiloride (10 ,uM) induced a mean hyperpolariza-tion of 24.6 ± 13 mV. Removal of Na+ from the externalmedium produced the same hyperpolarization as amiloride,and amiloride had no further effect in Na+-free medium (Fig.lAb). The amiloride derivative phenamil was more potentthan amiloride (in 140 mM Na+), whereas large concentra-tions of ethylisopropylamiloride (EIPA) (.100 ,M) wererequired to see an effect (Fig. lAd). Apparent K0.5 valueswere 19 and 56 nM for phenamil and amiloride, respectively.Although LE cells were electrically coupled, stable cur-

rents could be recorded (Fig. 1B). Addition of amiloride (10,uM) produced a marked, reversible shift in the outwarddirection of the current recorded at -20 mV (Fig. lBa). Thiseffect of amiloride was again seen only with Na+ in theexternal solution (Fig. lBb). Amiloride and its derivativesphenamil and benzamil were again effective in reducing thecurrent (Fig. 1B c and d). The order ofpotency was benzamil

phenamil > amiloride>> EIPA with corresponding ap-parent K0.5 values of 14 nM, 19 nM, 90 nM, and 0.4 mM.Similar results were obtained on currents recorded at -60mV (data not shown).Experiments were then performed to study single chan-

nels. Amiloride-sensitive NaCh were observed in 17 of 116outside-out patches. In asymmetrical conditions with K+ inthe pipette and Na+ in the bath, inward deflections ofcurrents were observed up to 50 mV. At higher potentials, nooutward current could be detected up to 100 mV (Fig. 2A).These results indicate a high selectivity for Na+ with a

A__________+40MV V(M'V)

C__- OmlaV -150 100 -50 5 1

C-.0- -40OmV K' 44 -~0.4C-0- somV Na+ g404 N( 0.2lpAfl 70mM Na 000 -100 -0d2V(V

35mM NA 0.8- V(mV)B .4

CONTROL AMILORIDE AMILORIDE WASHa O.iPM 1olaM

CONTROL 4000 AMILORIDE 23000P0PI.99 O.1PM0.I I~~ ~ Po=0.23 ] s

LiJ

AMILORIDE 17000101LM

Po=0.04

- 85

_.-0.5i

i(pA)

i(pA)WASH 12000Po=0.89

-0.5 0i(pA)

FIG. 2. Single-channel properties of the amiloride-sensitiveNaCh. (A) (Left) Single-channel current traces recorded at differentpipette potentials from an outside-out patch. (Right) Mean I-Vrelationships measured with different external Na+ concentrationsand K+-containing solution in the pipette. (Inset) I-V relationshipmeasured in Na+ symmetrical condition from two different outside-out patches. (B) (a) Long-lasting outside-out patch recording at -100mV, showing the blocking effect of amiloride. (b) Correspondingamplitude histograms from which P. values have been calculated.

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PNa*/PK+ ratio> 10. In Na+ symmetrical conditions the I-V bp). It contained a 2007-bp open reading frame encoding a

relationship crossed the voltage axis near 0 mV. The unitary 669-amino acid protein with a predicted size of 76 kDa (Fig.conductance was 4.4 pS from 100 mV toO0 mV. The activity 3). The Kyte and Doolittle hydrophobicity analysis (24)of this channel was also characterized by very slow kinetics indicates the presence of two large hydrophobic domains

with opening and closing times in the range of seconds. This (from amino acid 85 to 135 and 542 to 585). There are seven

channel was highly sensitive to amiloride (Fig. 2B). motifs ofN-glycosylation and a number of consensus sites for

Amiloride (100 nM) caused a 77% decrease in Po (n = 5). phosphorylation including one site for protein kinase A, five

Cloning and Ammno Acid Sequence of Human Lung NaCh sites for protein kinase C, and nine sites for casein kinase II.

(HLNaCh). A human lung cDNA library was prepared and Northern Blot Analysis and Ontogenesis of NaCh. The

screened with a fragment of the rat colon NaCh cDNA (20). mRNA for NaCh was specifically detected in human as well

Five positive clones were identifi'ed with sizes ranging from as in rat Na+-reabsorbing epithelial tissues (Fig. 4).- In

2.7 to 3.8 kb. They were transcribed in vitro and injected into humans, a 3.8-kb messenger was present in low abundance in

Xenopus oocytes. One expressing clone was sequenced (3151 pancreas but was expressed at a higher level in thyroid and

ccggccagcgggcgggctccccagccaggccgctgcacctgtcagggaacaagctggaggagcaggaccctagacctctgcagcccataccaggtctc -1

rat K F K P C P Q G

humnM E G N K L E E Q D S S P P S T P G L M K G N K R E E G 30

ATG GAG GGG AAC AAG CTG GAG GAG CAG GAC TCT AGC CCT CCA CAG TCC ACT CCA GGG CTC ATG AAG GGG AAC AAG CGT GAG GAG CAG GGG 90

rat S R *0hum L G P E P A A P 0 0 p T A E E E A L I E F H R S Y R E L F E 60

CTG GGC CCC G~AA~ GCG GCG CCC CAG CAG CCC ACG GCG GAG GAG GAG GCC CTG ATC GAG TTC CAC CGC TCC TAC CGA GAG CTC TTC GAG 180

rat 99K

hum F F C ~ T TT I H G A I R L V C S Q H N R K T A F A V L 90

TTC TTC TGC AAC AAC ACC ACC ATC CAC GGC GCC ATC CGC CTG GTG TGC TCC CAG CAC AAC CGC ATG AAG ACG GCC TTC TGG GCA GTG CTG 270

rat A E L

hum W L C T F G Y 0 F G L L F G FR Y F S Y P V S L N I N L 120

TGG CTC TGC ACC TTT GGC ATG ATG TAC TGG CAA TTC GGC CTG CTT TTC GGA GAG TAC TTC AGC TAC CCC GTC AGC CTC AAC ATC AAC CTC 360

rat 0 V T

hum N S K L V F P A V T I C T L N P Y R Y P E I K E E L E E L 150

AAC TCG GAC AAG CTC GTC TTC CCC GCA GTG ACC ATC TGC ACC CTC AAT CCC TAC AGG TAC CCG GAA ATT AAA GAG GAG CTG GAG GAG CTG 450

rat *N S Y R A *

humOD R I T E T L F L Y K Y S S F T T L V A G S R SO..R SR L 180

GAC CGC ATC ACA GAG CAG ACG CTC TTT GAC CTG TAC AAA TAC AGC TCC TTC ACC ACT CTC GTG GCC GGC TCC CGC AGC CGT CGC GAC CTG 540

rat L A F T Y S G T G s * V

hum R G T L P H P L R L R V p p p p H G A R R A R S V A S S L 210

CGG GGG ACT CTG CCG CAC CCC TTG CAG CGC CTG AGG GTC CCG CCC CCG CCT CAC GGG GCC CGTCA GCC CGT AGC GTG GCC TCC AGC TTG 630

rat Rhum R N N P V W K W K I G F L C N Q K S D C F Y T 240

CGG GAC AAC AAC CCC CAG GTG GAC TGG AAG GAC TGG AAG ATC GGC TTC CAG CTG TGC AAC CAG AAC AAA TCG GAC TGC TTC TAC CAG ACA 720

rat *S S A

hum Y S S G V A V R E W Y R F H Y I N I L S R L P E T L P S L 270

TAC TCA TCA GGG GTG GAT GCG GTG AGG GAG TGG TAC CGC TTC CAC TAC ATC AAC ATC CTG TCG AGGCG CCA GAG ACT CTG CCA TCC CTG 810

rat E A T A p 9K

hum E E T L G N F I F A C R F N V S C N Q A Y S H F H H P 300

GAG GAG GAC ACG CTG GGC AAC TTC ATC TTC GCC TGC CGC TTC AAC CAG GTC TCC TGC AAC CAG GCG AAT TAC TCT CAC TTC CAC CAC CCG 900

rat *4V

hum M Y G N C Y T F N K N S N L W S S P G I N N G L S L 330

ATG TAT GGA AAC TGC TAT ACT TTC AAT GAC AAG AAC AAC TCC AAC CTC TOG ATG TCT TCC ATG CCT GGA ATC AAC AAC GGT CTG TCC CTG 990

rat T T

hum M L R A E Q N F I P L L S T V T G A R V V H G D E P A 360

ATG CTG CGC GCA GAG CAG AAT GAC TTC ATT CCC CTG CTG TCC ACA GTG ACT GGG GCC CGG GTA ATG GTG CAC GGG CAG GAT GAA CCT GCC 1080

rat 0 A S N

hum F G G F N L R P G V E T S I S R K E T L D R L G G 390

TTT ATG GAT GAT GGT GGC TTT AAC TTG CGG CCT GGC GTG GAG ACC TCC ATC AGC ATG AGG AAG GAA ACC CTG GAC AGA CTT GGG GGC GAT 1170

rat E K

hum Y G C T K G VP V E N L Y P S K Y T V C I H S C 420

TAT GGC GAC TGC ACC AAG AAT GGC AGT GAT GTT CCT GTT GAG AAC CTT TAC CCT TCA AAG TAC ACA CAG CAG GTG TGT ATT CAC TCC TGC 1260

rat N K K K G F 0

hum F E S M I K E C G C A Y I F Y P R P N V E Y C Y R K H 450

TTC CAG GAG AGC ATG ATC AAG GAG TGT GGC TGT GCC TAC ATC TTC TAT CCG CGG CCC CAG AAC GTG GAG TAC TGT GAC TAC AGA MAG CAC 1350

ratf0G A L S

hum S S W G Y C Y Y K L Q V F S S H L G C F T K C R K P C S 480

AGT TCC TGG GGG TAC TGC TAC TAT AAG CTC CAG GTT GAC TTC TCC TCA GAC CAC CTG GGC TGT TTC ACC AAG TGC CGG MAG CCA TGC AGC 1440

rat I N K K D I E L

hum V T S Y L S A G Y S R P S V T S Q E W V F Q M L S R N 510

GTG ACC AGC TAC CAG CTC TCT GCT GGT TAC TCA CGA TGG CCC TCG GTG ACA TCC CAG GAA TOG GTC TTC CAG ATG CTA TCG CGA CAG AAC 1530

ra t4IL

humS Y T V N N K R N V A K V N I F F K E L N Y K T N S E S P 540

AAT TAC ACC GTC AAC AAC AAG AGA AAT OOA OTO GCC AAA GTC AAC ATC TTC TTC AAG GAG CTG AAC TAC AAA ACC AAT TCT GAG TCT CCC 1620

rat S

hum S V T V T L S N L G S 0 S L F G S S V L S V V A 570

TCT GTC ACG ATG GTC ACC CTC CTG TCC AAC CTG GGC AGC CAG TOG AGC CTG TGG TTC GGC TCC TCG GTG TTG TCT GTG GTG GAG ATG GCT 1710

rat I T L A

hum F L V F D L.L V T F L L L R R F R S R Y S P G R G R 600

GAG CTC GTC TTT GAC CTG CTG GTC ATC ATG TTC CTC ATG CTG CTC CGA AGG TTC CGA AGC CGA TAC TOG TCT CCA GGC CGA GGG GGC AGG 1800

rat R P F T PPP P 0

hum G A 0 E V A S T L A S S P P S H F C P H P M S L S L S P G 630

GGT GCT CAG GAG GTA 0CC TCC ACC CTG GCA TCC TCC CCT CCT TCC CAC TTC TGC CCC CAC CCC ATG TCT CTG TCC TTG. TCC CAG CCA GGC 1890

rat M T P L S A P L A P

hum P A P S P A L T A P P P A Y A T L G P R P P G G S A G A S 660

CCT OCT CCC TCT CCA 0CC TTG ACA 0CC CCT CCC CCT 0CC TAT 0CC ACC CTG GGC CCC CGC CCA TCT CCA GGC TCT GCA 0CC AGT 1980

rat C A A A A L

hum S S T C P L P

TCC TCC ACC TOT CCT CTG CCC tgagagggaaggagaggtttctcacaccaaggcagatgctcctctggtgggagggtgctggccctggcaagattgaaggatgt 2090

gcaggcttctccagaccgccaactgcgttgtgttggagggagcagatggtagggccaggagtgctcaagacagagctatgagctcccaaaggc 22101

tggcccagccttacccttgtacgccttgaaacttggttcccaccaactcggtaagctcttttccttgatagccagcgaactggactttacagg 23303ctttctaaaaacgctataacagacaaacacaccagggacagcagcatgacggtttctgccaggacgcttagccgccccgatggctggcacatg 24505ctccgtagacaatgttgctctctcttaactggggggaacccaccaaaacccctttttactagcaatcccttcctgaccccagggtaggctaag 25707

gaccgggtagtaaggagaccaggctctctacctataccgtccctacaggcctgcccggcccttgcctgttcttcatactcacattctgttgga 26909

aa 3052

FIG. 3. Nucleotide sequence of HLNaCh cDNA and the corresponding protein and alignment with the rat colon NaCh. In the rat sequence,only divergent residues are shown. *, Putative conserved N-glycosylation sites; o, phosphorylation sites by kinase A; o, phosphorylation sitesby kinase C; *, phosphorylation sites by casein kinase II.

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eE 0 XoU

_ n Q _

L -O=1 2

kb kb

3 Xl -

Human Ratco : days hours daysC_cc 7 -3 -1 1 +1 +20+60

kb _kb

-3.6

34

Humanlung

1.8

Rat lung

FIG. 4. Distribution of NaCh analyzed by Northern blot exper-iments. (A) Human tissues [2 ug of poly(A)+ except for thyroid (4Mg)] and rat tissues (6 pg of total RNA). FLE, fetal LE cells. (B)Human lung [1.2 yg ofpoly(A)+], fetus (20-25 weeks), and adult. (C)Rat lung (6 Mg of total RNA). (Upper) Hybridization with rat colonNaCh probe. (Lower) Control hybridization with a ,-actin probe.Days before birth (-) and after birth (+).

even more in human kidney. This same mRNA was found inlung and colon. In these two tissues, another 3.4-kb transcriptwas also observed (Fig. 4A).A large increase of expression of NaCh was found from

fetal to adult stage in human lung (Fig. 4B). During devel-opment of rat lung (Fig. 4C), NaCh mRNA appeared a fewdays before birth, with a large increase in expression aroundthe time of birth. The level of expression remained high in theadult stage. NaCh mRNA was also detected in a primaryculture of LE cells used for electrophysiology (Fig. 4A).Human Chromosome Localization. In the 100 metaphase

cells examined after in situ hybridization, there were 184silver grains associated with chromosomes, and 63 of these(34.2%) were located on chromosome 12. The grain distri-bution on this chromosome was not random; 50/63 (79.4%)mapped to the p13 band of the short arm of chromosome 12(Fig. 5).

Functional Expression of the HLNaCh in Xenopus Oocytes.Oocytes injected with cRNA expressed an amiloride-sensitive current in 98 mM Na+ medium (Fig. 6 Inset, traceA). When external Na+ was replaced by K+ (trace B), noamiloride-sensitive current was observed. Fig. 6 gave Ko.5values of 50 and 80 nM for inhibition by phenamil andamiloride, respectively. Ethylpropylamiloride (EPA) at 10,uM had no effect on NaCh activity.

342" 1e@ee@ O@eeeeeeOeee-@@ @@

1 2q,3 -

.......... ....0

12

FIG. 5. Idiogram ofhuman G-banded chromosome 12 illustratingdistribution of labeled sites for the HLNaCh probe.

4- 100 i

amilonde~5O

x 50 1 : X *amilorideE 0 phenamil0 ~~------ B AEPA

0-/-0O .9 .7 -5

log [diuretic]FIG. 6. Electrophysiological and pharmacological characteriza-

tion of HLNaCh in Xenopus oocytes. Dose-response curves foramiloride and its derivatives phenamil and EPA (n = 4 for eachcompound). (Inset) Effect of 20 ,uM amiloride on HLNaCh cRNAexpression. Trace A, reversible effect of amiloride in 98 mM Na+;trace B, selectivity for Na+ measured in a Na+-free buffer (replace-ment of Na+ by K+).

DISCUSSIONThe existence of amiloride-sensitive conductive pathways inalveolar epithelial cells has been demonstrated by macro-scopic measurements (3-6). Patch-clamp studies have iden-tified an amiloride-sensitive Ca2+-activated nonselectivechannel (PNa+/PK+ = 0.9) in fetal alveolar epithelial cells (16).However, because of the high intracellular Ca2+ concentra-tion required to activate this channel (25), its role in Na+absorption seems difficult to explain.The electrophysiological data presented in this paper show

that the NaCh in LE cells has a conductance of 4 pS, a highselectivity for Na+ versus K+, slow kinetics, a relatively highaffinity for its blocker amiloride (Ko.5 = 90 nM) and foramiloride derivatives such as phenamil and benzamil, and aquasi-insensitivity to the Na+/H+ exchange blocker EIPA(26). These properties are very similar, if not identical, tothose described for the amiloride-sensitive NaCh in the renalcollecting tubule (12, 27).The cloned human lung NaCh is a 76-kDa protein with a

structure similar to that established for the rat colon NaCh(20, 28), with which it has 81% identity if Met-27 of the ratcolon structure (20) is aligned with the first methionine(Met-1) of the human sequence.Labeled [3H]benzamil (9) and [3H]bromobenzamil (10)

have been used in an attempt to biochemically characterizethe amiloride-sensitive NaCh in pneumocytes. The corre-sponding binding sites have properties similar to those oftworecently cloned amiloride binding proteins (ABPs) (29).Transfected cells expressing long or short ABPs expressedhigh levels of binding for amiloride and amiloride derivativesbut did not express NaCh activity (29). There is no structuralhomology between the HLNaCh cloned in this work and theABPs.Both the ionic selectivity and the pharmacology of HL-

NaCh expressed inXenopus oocytes are very similar to thosefound by patch-clamp techniques on cultured pneumocytes.The cloned channel is Na+ selective versus K+ and its K0.5values for amiloride (80 nM) and for phenamil (50 nM) arevery similar to those observed in intact pneumocytes. Thechannel is essentially insensitive (up to 10 AM) to EPA, anamiloride derivative that blocks Na+/H+ exchange.For all these reasons, there is little doubt that the cloned

protein is an essential element of the NaCh structure. How-ever, the relatively low intensity of the expressed amiloride-sensitive Na+ current observed after injection of HLNaCh

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cRNA into oocytes (50-100 nA) might suggest that othersubunits are required for generating a higher level of activity,as previously observed for other ionic channels (30). Theseother subunits might participate in Na+ permeation or, alter-natively, might be components necessary for a proper mat-uration of the cloned protein.Two distinct forms of transcripts were detected in human

tissues at 3.4 and 3.8 kb. Expression in lung, kidney, andcolon has very similar characteristics. The case of the thy-roid, which expresses only the 3.8-kb messenger, might bedue to the fact that, in this tissue, the amiloride-sensitiveNaCh is different. It has a low Na+ versus K+ selectivity (31).The expression of HLNaCh is highly regulated during

development. The channel mRNA is not expressed at thefetal stage (20-25 weeks) but is, as expected, fully expressedin adults. This observation is to be linked to the fact that fetallung behaves as a Cl--secreting epithelium and adult lungbehaves as a Na+-reabsorbing one. A more detailed analysisof NaCh ontogenesis in the rat confirmed that expression ofmRNA increased considerably just before birth. Becausesteroids are well-known regulators of amiloride-sensitiveNaCh function in kidney (11, 32) and colon and becausecorticosteroids play a role in terminal lung differentiation(reviewed in ref. 33), steroids appear as excellent potentialcandidates for transcriptional regulators during lung devel-opment.One particularly important aspect of the lung NaCh is its

involvement in cystic fibrosis. The disease is now well knownto be associated with mutations ofCFTR, a cAMP-dependentCl- channel (34). However, while the CFTR Cl--channelactivity is drastically reduced for the most severe mutationssuch as I?he-508 deletion (18), NaCh activity, in the mean-time, is significantly increased (14). The combination of thetwo abnormalities results in a severe modification of thewater balance that is the central feature of the disease. Aconsequence of the increased functional expression ofNaChin cystic fibrosis is that aerosol administration of amiloridehas been proposed as a treatment of the disease (15). It isprobably important to know in that respect that the HLNaChhas the same pharmacology as kidney and colon NaCh witha particularly high affinity for amiloride.Numerous human diseases are associated with channel

function (reviewed in ref. 35). It would not be surprising thatmutations of NaCh are also associated with diseases inrelation to transepithelial Na+ movements. This work pro-vides probes to analyze the possible implications of thischannel type in genetic diseases and indicates that theHLNaCh gene is localized on chromosome 12.

N.V., E.L., and G.C. contributed equally to this work. We aregrateful to S. Renard for discussions and for very useful help indifferent aspects of this work and to Prof. Jose Santini (H6pitalPasteur, Nice) for human thyroid biopsies. We thank F. Aguila, V.Friend, C. Le Calvez, R. Pichot, and C..Roulinat for expert technicalassistance. This work was supported by the Association Frangaise deLutte contre la Mucoviscidose, the Centre National de la RechercheScientifique, and the Institut National de la Sante et de la RechercheMedicale (Grant 91.0204).

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