recent advances in the molecular understanding of voltage-gated ca2+ channels

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MCNMolecular and Cellular Neuroscience 14, 255–272 (1999)

Article ID mcne.1999.0795, available online at http://www.idealibrary.com on

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EVIEWecent Advances in the Molecular Understandingf Voltage-Gated Ca 21 Channels

ndrew Randall1 and Christopher D. Benham
epartment of Neuroscience Research, SmithKline Beecham Pharmaceuticals, New Frontierscience Park, Harlow, Essex, CM19 5AW, United Kingdom
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NTRODUCTION

It has long been recognized that the activity ofoltage-sensitive Ca21 channels (VSCCs) is central to theunction of all excitable cells (i.e., cells that fire actionotentials), as well as many nonexcitable cells (Hille,992). VSCCs provide a crucial link between a cell’sembrane potential and the enormous number of intra-

ellular processes that either directly utilize elevationsf [Ca21]i as a functional trigger (e.g., exocytosis, muscleontraction) or are modulated by Ca21-dependent signal-ing cascades (e.g., gene expression, cell division) (Catter-ll, 1995; Bito et al., 1997; Morgan and Curran, 1988;anabe et al., 1993; Dunlap et al., 1995).The cloning and molecular characterization of VSCC

ubunits has allowed many crucial insights into theirtructure, physiology, pharmacology, and potential roles inisease. As we approach the end of the millennium ourrevious understanding of VSCCs has been both enhancednd challenged by the molecular identification of a host ofew VSCC subunits. These include three subunits that giveise to members of the hitherto highly elusive T-type (orow-voltage activated) Ca21 channel family.

Driven by molecular cloning and mutational analysis,ur understanding of how ion channel structure relateso function is growing apace. Along with this, andupported by the sequencing of the human genome, were now gaining increasingly numerous novel insightsnto how ion channel mutations underpin a range ofathological states. Many excellent and substantial over-iews of the properties, genetics, functions, and diver-ity of VSCCs have been produced, some recent ex-

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1 To whom correspondence and reprint requests should be ad-ressed. E-mail: [email protected].

044-7431/99 $30.00opyright r 1999 by Academic Pressll rights of reproduction in any form reserved.

mples of which include Catterall (1995); Tsien andheeler (1999); Snutch and Reiner (1992); Dolphin

1998); Chin (1998); Ophoff et al. (1998); Hofmann et al.1994); Randall (1997); Walker and De Waard (1998); anden (1999). In this brief review we will concentrate onhree topics in which important advances have been

ade recently. First, we discuss how the recent cloningf a number of new VSCC subunits both changes andnhances our view of Ca21 channel diversity. Followinghis, we will describe the emerging data on the identi-ed roles of Ca21 channel subunit mutations in disease.inally, we will describe recent work that has character-

zed a major molecular mechanism by which intracellu-ar Ca21 levels can produce feedback modulation of thectivity of VSCCs.

RIOR ART IN THE MOLECULARIOLOGY OF VSCCs

The first complement of VSCC subunits were clonedrom skeletal muscle in the late 1980s (Tanabe et al., 1987;uth et al., 1989; Ellis et al., 1988; Jay et al., 1990), workade possible by a preceding period of protein chemis-

ry and purification. In the following years the genes forconsiderable number of additional VSCC subunits and

heir splice variants were isolated, predominantlyhrough homology-based cloning (Mikami et al., 1989;

ori, 1991; Dubel et al., 1992; Starr et al., 1991; Williams et al.,992a, 1992b, 1994; Soong et al., 1993; Hullin et al., 1992;astellano et al., 1993a, 1993b; Perez-Reyes et al., 1992).These studies provided a substantial increase in our

nderstanding of the structure of VSCCs and haveubsequently permitted a broad range of novel biologi-
al investigations to be performed. Thus, within aecade of the isolation of the first VSCC gene, topics as
255

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256 Randall and Benham

iverse as control VSCC selectivity by single aminocids to the identity of channel mutations causingnheritable human disease states have been studied.

VSCCs are thought to exhibit the heteromeric struc-ure illustrated in Fig. 1 (Walker and De Waard, 1998;atterall, 1995). In skeletal muscle, the central pore-

orming a1 subunit is associated with a predominantlyxtracellular a2d subunit (the disulfide-linked productf a single gene), an intracellular b subunit, and a

IG. 1. A schematic representation of the heteromeric assembly pomologous repeats of the pore-forming a1 subunit (red) with a cut-oelix and S2-S6 purple columns). The loop between S5 and S6 dips inssociated with the a1 subunit are the entirely intracellular b subunitd subunit (green). This latter subunit pairing consists of the single
2
xtracellular a2 subunit. The well-characterized sites of contact between a1 af the AID/BID sites and additionally through an interaction between the tw

embrane spanning g subunit. All other calcium chan-els including those in neurones, endocrine cells, andmooth and cardiac muscle were thought to have aimilar heteromeric structure, except for the lack of a gubunit. The basic physical properties of each of theseSCC subunits are outlined in Table 1.a1 Subunits are the primary determinants of both

a21 channel biophysics and pharmacology. These largeour motif subunits form the pore (Yang et al., 1993;

n and topology of an a1a2dbg VSCC. The figure illustrates the 4view of repeat I showing the 6 putative transmembrane a-helices (S1e membrane to form the lining of the pore (see arrowhead in pore).

ge), the four times membrane traversing g subunit (sky blue), and thesmembrane crossing d subunit disulfide linked (S–S) to the entirely

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Recent Advances in Molecular Understanding of VSCCs 257

irksen et al., 1997), the voltage sensor(s), and thectivation and inactivation ‘‘gates’’ (Nakai et al., 1994;arcia et al., 1997; Zhang et al., 1994). In addition, a1

ubunits contain regions responsible for modulatorynteractions with G-proteins (Dolphin, 1998) and othershat form the binding sites for all well-characterizeda21 channel antagonists (Mitterdorfer et al., 1998;triessnig et al., 1998; Ellinor et al., 1994).As such, a1 monomers can be considered the pheno-

ypic heart of all VSCCs. Indeed, this subunit aloneontains all that is required to produce a fully functionaloltage-gated calcium channel and in many cases isapable of producing functional channels when ex-ressed in isolation. Nevertheless, many, if not all, Ca21

hannels are heteromultimers in vivo (Fig. 2). The b, a2d,nd g subunits that combine with a1 in these hetero-eric complexes are best regarded as regulators orodulators of VSCC function. However, as demon-

trated both by subunit coexpression studies and tar-eted gene knockouts, the role of these accessory sub-nits in shaping VSCC function is clearly important.A combination of biophysical and pharmacological

pproaches has been used to differentiate a number ofifferent classes of VSCC in native cells. Currently thelassification these native VSCCs uses an extension ofhe L-, N-, T-type nomenclature first adopted by Tsiennd colleagues (Fox et al., 1987; Nowycky et al., 1985).hrough the subsequent addition of the P-, Q-, and

ABLE 1

asic Properties of VSCC Subunits

Mol. Weight (kDa) a1

redicted TM segments 24 1 (in d)lycosylation sites Yes Yeshosphorylation sites Yes Norug interactions Numerous inc. dihy-

dropyridines phenylal-kylamines invertebratetoxins

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a1

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pathway, selectivitymechanism, voltagesensor, and inactivationgate

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-type channels, the native VSCC family currentlyxtends to six members (Llinas et al., 1992; Mintz et al.,

SP

992; Randall and Tsien, 1995; Zhang et al., 1993; Randallnd Tsien, 1997b). One important challenge of recentears has been to match the properties of these nativehannels with those of recombinant VSCCs (Randall,997a; Tsien and Wheeler, 1999; Zhang et al., 1993).By the middle of 1994, it seemed possible that all theajor VSCC had been isolated subunits from the mam-alian genome. Recombinant a1 subunits that encoded

hannels akin to most of the native VSCCs had beensolated. The only major doubt concerned the moleculardentity of the T-type channel. Some (Bourinet et al.,996; Soong et al., 1993), but not others (Williams et al.,994; Randall and Tsien, 1995; Zhang et al., 1993; Waka-ori et al., 1994), thought the properties of T-type

hannels were conferred by the presence of the a1Eubunit. Another report claimed that the T-type chan-els represented a1 subunits functioning in the absencef b and a2d subunits (Meir and Dolphin, 1998) (Fig. 2).Another irksome question concerned the precise mo-

ecular identity of the native channels designated P- and-type. Currents carried by these two channel types coulde resolved both biophysically and pharmacologicallyhrough careful analysis of the Ca21 currents of native cellsRandall and Tsien, 1995). Both P- and Q-type currentsLlinas et al., 1992; Mintz et al., 1992; Randall & Tsien, 1995),owever, bore some resemblance to the currents producedy expression of the recombinant a1A VSCC subunit (Mori,991; Sather et al., 1993; Berrow et al., 1997; Niidome, 1994;

b g

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tea et al., 1994). Consequently, a1A was assigned to the-type channel, the Q-type channel, or, more commonly,

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oth. Several resolutions to this issue were therefore pos-ible. It was conceivable that one or more P- or Q-type Ca21

hannel-encoding a1 subunits remained uncloned. Alterna-ively, the differences between P- and Q-type VSCCs couldave arisen from alternative splicing of a1A, the presence orbsence of different a2d, b, or g subunits, or some form ofosttranslational modification to the a1 or any other VSCCubunits.

As described below recent advances seem to haveesolved both the T-type and P/Q-type issues and nowrovide a seemingly more complete picture of theatches between recombinant and native VSCCs.

WAVE OF NEW VOLTAGE-SENSITIVEALCIUM CHANNEL SUBUNITS

IG. 2. Possible subunit partnerships for VSCC. The figure illustrateskeletal muscle channels are thought to typically consist of the a1S, b1

lsewhere consist of one of the five other HVA-specific a1 subunits (i.ehannels outside of skeletal muscle may contain g2 or g3. The subunit chey certainly contain an a1 subunit (either a1G, H, or I), which alorobably lack b subunits. The presence or absence of either a2d or g su

Recently, after a hiatus of some four years in which noajor new genes were isolated, new cloning efforts,

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redominantly triggered by bioinformatics, have lead tohe isolation of genes for a surprising number ofew VSCC subunits (Fig. 3). Most notable among

hese products of the in silico cloning era are threeenes that encode bona fide T-type Ca21 channels a1ubunits (Cribbs et al., 1998; Perez-Reyes et al., 1998;ee et al., 1999b; Williams et al., 1999; Klugbauert al., 1999b; Mittman et al., 1999). In addition re-ent work has uncovered a new L-type calcium channelene specific to the retina (Strom et al., 1998; Bechhansent al., 1998), new members of the a2d subunit familyKlugbauer et al., 1999a) and VSCC g subunit paralogueshat are expressed in locations other than skeletal

uscle (Letts et al., 1998; Black and Lennon, 1999),ncluding the CNS. Below we will expand onhese recent advances in our understanding of Ca21

hannel heterogeneity and will discuss how they may

ost likely subunit combinations exhibited by different VSCC classes.1, and g1 subunits. High-voltage activated (HVA) channel assemblies, B, C, D, or E) plus an a2d subunit and a b subunit. In addition HVA

osition of low-voltage activated (LVA) channels is currently less clear.oduce functional channels with suitable properties. In addition theys is either contentious or remains to be rigorously tested.

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loning of Three T-Type Ca 21 Channel a1

ubunit Genes

The most important recent additions to the phenotype-efining a1 VSCC subunit family have been the subunitsnown as a1G, a1H, and a1I. The expression of each ofhese three subunits, in either Xenopus oocytes or HEK93 cells, produces low-voltage activated (LVA), slowlyeactivating whole cell currents and small conductance

,8 pS) single channel currents (Cribbs et al., 1998;erez-Reyes et al., 1998; Lee et al., 1999b; Williams et al.,999; Klugbauer et al., 1999b). The combined presence ofhese biophysical hallmarks allow us to indisputablylassify a1G, a1H, and a1I as subunits that encode T-typea21 channels (Huguenard, 1996; Kostyuk, 1999; Tsien et

l., 1998).Although the channels produced by expression of a1G,

1H, and a1I all share the classic biophysical hallmarks of-type VSCC family, they also exhibit significant indi-idual differences. This is not surprising given that theverall level of sequence homology among these threeubunits lies around 70%. A similar level of sequenceivergence is seen in other ion channels that displayeasurable functional and pharmacological diversity;

or example, the different high-voltage-activated (HVA)a21 channels. A good example of the functional diver-

ity within the T-type channel group lies in the particu-arly slow activation and inactivation kinetics of cur-ents mediated by a1I subunits when compared withhose produced by a1G or a1H subunits (Lee et al., 1999b).nother example, is the as-great-as-40-fold difference in

IG. 3. A timeline of VSCC subunit cloning. The graph illustrates theSCC subunits. Success in these endeavors can be divided into three m

rom skeletal and cardiac muscle. These successes relied on access to larnd obtain sequence information. The second cloning epoch (the brauscle era to homology-clone channels largely expressed in the CNS

earching algorithms and large-scale sequencing efforts are combineharacterized VSCC subunits.

he Ni21-sensitivity of the three different T-type channelubunits, a result that points to diversity within the

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ermeation pathway of LVA VSCCs (Lee et al., 1999a).hese differential biophysical properties of the three-type channel a1 subunits go some way to explaininghe range of LVA VSCC phenotypes that have beenreviously reported in native cells (Huguenard, 1996;ostyuk, 1999; Akaike, 1991; Tarasenko et al., 1997;huravleva et al., 1999; Tsien et al., 1998).Full-length a1G and a1I have been cloned from both

odent and human tissues (Perez-Reyes et al., 1998; Leet al., 1999b; Klugbauer et al., 1999b), whereas a1H hasnly been isolated from human (Williams et al., 1999;ribbs et al., 1998). There are splice variants of all three

hannels. As might be expected from both their distinc-ive biophysics and their longstanding resistance toomology cloning efforts, sequence comparisons be-

ween the T-type channel genes a1G, a1H, and a1I and theVA channel a1 subunit genes indicate substantialifferences. The overall predicted topology of the T-typehannel, however, is identical to that of both the HVASCCs and Na1 channels. Moreover, the regions of the

hannel known to be important in making ‘‘a VSCC aSCC,’’ most notably the transmembrane helices andore forming loops, are well conserved across the HVAnd LVA VSCC families.The distribution of T-type Ca21 channel a1 subunitRNAs is commensurate with prior electrophysiologi-

al reports of T-type Ca21 currents in both neuronal andonneuronal tissues. a1G and a1I subunits are expressedredominantly, but not exclusively, in the CNS, withoth genes being expressed at relatively low levels in

of the first published reference to the cloning of each of the 20 differentepochs. In the first (the muscle era, 1987–1989), channels were cloned

ounts of tissues and the consequent ability to purify channel proteinsa, 1990–1994) used nucleotide sequence information obtained in thee most recent epoch (the in silico cloning era 1998–present), databaseaid identification of channels with low homology to the previously

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he testis and lung, for example (Talley et al., 1999; Kaset al., 1999; Klugbauer et al., 1999b). In contrast, a1H is

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xpressed at high levels in many peripheral tissues,ncluding heart, kidney, and pituitary, as well as in theNS (Williams et al., 1999; Cribbs et al., 1998; A. D.edhurst, personal communication). Within the brain,

he three different T-type VSCCs a1 subunits exhibitifferential mRNA distributions, although significantverlap of their sites of expression is also seen (Talley etl., 1999). Of the three subunits a1G appears to bexpressed at the highest levels in the CNS. The distribu-ion and biophysics of this channel suggest that it isrimarily responsible for the classic thalamic T-typea21 current (Huguenard, 1996).The interactions of a1G, a1H, and a1I with other VSCC

ubunits is not yet well characterized. When expressedlone, in either oocytes or mammalian cells, all three-type Ca21 channel a1 subunits thus far cloned pro-uce robust LVA currents (Cribbs et al., 1998; Lee et al.,999b; Perez-Reyes et al., 1998; Klugbauer et al., 1999b;illiams et al., 1999). This suggests that the a1 G, H, and

subunits can function adequately in the absence of a2d,, and g subunits (Fig. 3). Indeed the current amplitudesroduced are far in excess of those observed when anyVA VSCC is expressed alone. This result alone, how-

ver, does not rule out interactions with the VSCCccessory subunits. With regard to the a2d subunits,here are currently two conflicting results concerningnteractions with the LVA a1G subunit. Lacinova et al.1999) claim no interaction with either a2d-1 or a2d-3 (seeelow), whereas a subsequent study points towardome interaction with a1G, with current amplitudeseing increased around twofold by a2d-1 coexpressionDolphin et al., 1999), a result that mimics the effects of2d on HVA channels.None of the three T-type Ca21 channels cloned to date

xhibits the b subunit interaction site expressed by allVA VSCCs (Walker and De Waard, 1998; De Waard et

l., 1994; Pragnell et al., 1994). This suggests that theell-characterized actions of b subunits on VSCC activa-

ion and inactivation may be restricted to the HVA classf channels. This hypothesis, however, is yet to beigorously tested.

Both T-type channels and the newly identified nonskel-tal muscle VSCC g subunits (see below) are implicatedn the pathophysiology of epilepsy—particularly that ofbsence seizures (Letts et al., 1998; Black and Lennon,999). For this reason, the possibility of an interactionetween the LVA a1 subunits and brain-expressed gubunits is seductive. To date, no studies of this interac-ion have appeared in the literature; however, work byne of us (ADR, N. C. L. McNaughton and Paula Green)
nd at least one other group (E. Perez-Reyes, personalommunication) has as yet failed to uncover evidence
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or any substantial g subunit modulation of LVA chan-el function.

Novel Retinal L-Type Channel Gene

The HVA VSCC channel family can be divided intowo distinct subgroups: the L-type channels and theresynaptic Ca21 channels. The former group is definedy HVA biophysics and voltage-dependent antagonismy dihydropyridines such as nimodipine, nifedipine,nd amlodipine. The latter group can simply be defineds HVA and insensitive to dihydropyridines, but areabeled presynaptic here by dint of their role in fasteurotransmitter release at action potential-dependentynapses (Dunlap et al., 1995; Wu et al., 1998; Wheeler etl., 1994; Takahashi and Momiyama, 1993). The func-ional and pharmacological differences between the-type and presynaptic Ca21 channels are also reflected

n the VSCC phylogenetic tree, where each family formsseparate branch of the limb formed by the HVA

hannels (Fig. 4).Until recently the L-type Ca21 channel family has

een thought to consist of three members. The first, a1S,s a Ca21 channel specialized for driving excitation-ontraction coupling in skeletal muscle (Tanabe et al.,993). a1C is the L-type channel involved in the cardiacction potential but is also involved in other functionsncluding those of the CNS (Mikami et al., 1989). Finally,1D is the major L-type channel expressed in CNS where

ts roles probably include triggering Ca21-dependentene expression (Bito et al., 1997) and dendritic releasef peptides (Simmons et al., 1995). a1D is also found inany endocrine cells where it plays an important role in

timulus secretion coupling (Ashcroft et al., 1994; Bokvistt al., 1995). Lately, genetic mapping work has seeminglydded another channel, a1F, to the L-type channelrouping. The gene encoding this putative 1966 aminocid L-type channel is mutated in individuals with-linked congenital stationary night blindness (Strom etl., 1998; Bechhansen et al., 1998).

To date, no reports of the functional expression of the1F gene have been made, although its expression iseported to be limited to the retina. It is tempting topeculate that expression of a1F will produce L-typeurrents with the distinctive fast kinetics of the L-typehannels that drive neurotransmitter release at thection potential-independent ribbon synapses of retinalells (Mennerick and Matthews, 1998). Electroretinogra-hy and adaptometry on afflicted individuals indicate

hat the phenotypes produced by different inherited
utations in a1F vary in severity from reduced function
n the rod pathway to complete loss of rod-mediated

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ignalling. It may therefore be enlightening to character-ze the biophysical properties of these different natu-ally occurring a1F mutants.

ovel a2d and g Subunit Genes

Although the a1 subunits are the primary determi-ants of VSCC phenotype, the accessory subunits a2d, b,nd g modulate VSCCs in a range of important ways.he VSCC b subunit family has not expanded since theloning of its fourth member was reported in 1993Castellano et al., 1993a; Castellano and Perez-Reyes,994). In contrast, both a2d and g subunit families haveeen increased from single to multiple membership

IG. 4. Phylogentic relationships between VSCC a1 subunits. An unro1 subunits. Full open reading frames were aligned using CLUSTALWeries substitution matrix. Scale bar is a function of amino acid substituiewed using TREEVIEW [Page (1996) Comput. Appl. Biosci. 12: 357–358hannels, all of which are sensitive to the dihydropyridine VSCC anemonstrated to play some role in fast vesicular release at action pharacteristic low-voltage activated phenotype.

ithin the past 18 months.The a2d family has been expanded by the recent

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dentification of two novel subunits a2d-2 and a2d-3Klugbauer et al., 1999a). Sequence comparison of thehree a2d subunits reveals quite substantial differencesetween individual family members; the conservationetween a2d-2 and a2d-3 and a2d-1 being 56 and 30%,espectively. Despite this, analysis of hydrophobicityrofiles, the location of potential glycosylation sites andisulfide bridge-forming cysteines points to a strongtructural conservation across three a2d subunits. Fur-hermore, in coexpression studies, a2d-3 (like a2d-1) haseen shown to be functionally effective in modulatingurrents produced by both the a1C and a1E VSCCubunits (Klugbauer et al., 1999a). In contrast, functionalffects of a d-2 coexpression have yet to be demon-

dendrogram prepared from the amino acid sequence of human VSCCompson et al. (1994) Nucleic Acids Res. 22: 4673–4680] with the PAMscores based on the PAM series substitution weight matrix. The tree ise phenotype-defining channels fall into three clear groups: The L-typenists; the presynaptic VSCC subunits, all of which encode channelsial driven synapses; and the T-type channels, all of which exhibit a

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pread. In contrast, a2d-3 expression is concentratedithin the CNS (Klugbauer et al., 1999a). As a result of

heir considerable sequence divergence, it may be pos-ible to specifically target novel drugs to differentembers of the a2d family. Interestingly in this regard,

he anticonvulsant/antinociceptive agent gabapentininds to a2d subunits, and in tissue sections does so withbinding pattern that best reflects the localization of

2d-1 (Gee et al., 1996; Klugbauer et al., 1999a).

ovel Nonmuscle VSCC g Subunits

A popular consensus for many years was that theresence of the VSCC g subunit was restricted tokeletal muscle where the product of a single gene (g1)ssociates with the a1S subunit (Jay et al., 1990; Ludwig etl., 1997). This view was recently called into question byhe cloning of what appeared to be a VSCC g subunitrom murine brain (Letts et al., 1998). The g2 subunitalso known as stargazin) was isolated by identifyinghe locus of the genetic disruption in the epileptic mouse

utant lines known as stargazer and waggler. Although1 and g2 share a similar exon structure, the sequenceomology between them is modest (25% identity, 38%imilarity in 200 amino acids of overlapping sequence);urthermore g2 contains an 100 amino acid extension notresent in g1. Despite this, the hydrophobicity profilend single N-linked glycosylation consensus site of g2

rovide a close match with that determined for g1. Asuch both subunits are predicted to contain four trans-embrane regions, with g2’s carboxyl extension likely

o be exclusively intracellular (Letts et al., 1998).The isolation of g2 from mouse rapidly lead to the

solation of its human orthologue, a gene present onhromosome 22 (Black and Lennon, 1999). The isolationf human g2 also leads to the identification of andditional g subunit paralogue g3, which is located onuman chromosome 16 (Black and Lennon, 1999). These

wo human g subunits share structural homology withach other and with the mouse g1 and g2 subunits.omparable to the situation reported in the mouse,uman g1 and g2 exhibit only slight homology (18%). Aimilarly low level of homology is seen between human1 and g3 (17%). In contrast, human g2 and g3 both havehe long C-terminus first reported for mouse g2 andxhibit a much greater degree of sequence homology.iven that g2 was first isolated from seizure proneutant mice, another potentially important observation

s the mapping of human g3 to a region of humanhromosome 16p12 close to the locus for familial infan-
ile convulsions and paroxysmal choreoathetosis (Lee etl., 1998; Szepetowski et al., 1997).
st

Although a functional interaction between g2 anduscle L-type channels is widely accepted, there is still

onsiderable debate as to whether the newly cloned g2

nd g3 subunits really form part of native VSCCs. Toate, the only reported evidence linking the new gubunits to VSCC function is a subtle hyperpolarisinghift in the steady state inactivation curve of rabbit1A-mediated Ca21 currents produced by mouse g2

Letts et al., 1998). It remains to be seen if this effect isidely reproducible, applies to other VSCCs, or can be

bserved when species-matched subunits are examined.

ISSECTING THE MOLECULAR BASIS OF- AND Q-TYPE CHANNELS

The expression in vivo of the a1A VSCC subunit isenerally believed to produce the native currents desig-ated P- and Q-type. Indeed, this perceived commonolecular background has lead to a quite widespread

doption of the channel designation of P/Q-type, aombined class that encompasses channels that arenhibited by the spider toxin v-Aga-IVA (Llinas et al.,992; Mintz et al., 1992; Randall and Tsien, 1995). The usef this agent and other peptide toxins has revealed theresence of P/Q-type channels in many different classesf neurone, a result that is commensurate with theidespread expression of the a1A gene (Westenbroek et

l., 1995; Day et al., 1996, 1997; Craig et al., 1998).dditional pharmacological experiments have revealed

hat P/Q-type channels are crucially involved in rapideurotransmitter release at many excitatory and inhibi-

ory synapses (Takahashi and Momiyama, 1993; Wheelert al., 1996; Luebke et al., 1993).

When examined in different cell-types v-Aga-IVA-ensitive currents display a range of functional character-stics. Observations of this nature, along with compari-ons to the pharmacological and biophysical propertiesf expressed recombinant a1A subunits (Sather et al.,993; Niidome, 1994; Mori, 1991; Berrow et al., 1997;oreno et al., 1997; Stea et al., 1994), lead to the original

eparation of v-Aga-IVA-sensitive currents into distinct- and Q-type components (Randall and Tsien, 1995).he two main distinctions between these P- and Q-typeurrents lay in their rate of voltage-dependent inactiva-ion (very slow for P-type, moderate for Q-type) andheir sensitivity to v-Aga-IVA (IC50s of ,1 nM for P-typend ,0.1 µM for Q-type). Subsequent work on differentystems has revealed the presence of v-Aga-IVA-
ensitive currents with characteristics intermediate tohose of the classical P-type currents of Purkinje neu-

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ones and the classical Q-type currents of cerebellarranule cells.Some 6 years after the initial claims of separate P- and-type channels, analysis of a1A splicing patterns and

ccessory subunit interactions has seemingly uncoveredhe molecular basis of the P/Q distinction (Bourinet etl., 1999). Alternate splicing of the original form of theat a1A subunit (a1A-a) can produce the insertion of aaline at position 421 in the I-II linker, an asparagine–roline insertion in IVS3–IVS4 extracellular loop and anlternate EF hand region in the C-terminus. All three ofhese changes are seen in a a1A isoform denoted a1A-b.haracterization of the properties of these splice varia-

ions reveals a number of important differences.The insertion of Valine at position 421 converts

trongly inactivating currents, reminiscent of native-type channels, into noninactivating responses that

esemble the slow inactivation of P-type currents (Bouri-et et al., 1999). The slow inactivation of a1A-V421 isarticularly striking when coexpressed with b2a, a sub-nit, which like a1A, is highly expressed in cerebellarurkinje cells (Volsen et al., 1997), the neurone in whichlassical P-type currents were first described (Llinas etl., 1992; Regan, 1991). The asparagine–proline insertionn IVS3–IVS4 produces a lowering of v-Aga-IVA affinity,uggesting that its presence may generate the relatively-Aga-IVA-insensitive Q-type channel (Randall andsien, 1995).The work of Bourinet and colleagues demonstrates

hat a broad range of v-Aga-IVA-sensitive channelhenotypes can be produced by alternative splicing of

he a1A subunit. Their work goes much of the way toxplaining the phenotypic differences of the P- and-type channels first described by Randall and Tsien

1995). The generation of an a1A knockout mouse inhich both Purkinje cell P-type currents and granule

ell Q-type currents are lost (R. W. Tsien, personalommunication) supports the mediation of these twoative currents by products of the same gene. With thisew knowledge in hand, the time may be approaching

o reassess the nomenclature of native a1A-mediatedSCCs.

ALCIUM CHANNEL MUTATIONSND DISEASE

In the past, the only clinical conditions associatedith mutations in VSCC genes were disorders of skel-

tal muscle. The best characterized of these resulted
rom alterations in the muscle-specific dihydropyridine-ensitive a1S subunit. Mutations in this gene produce a
sm

ange of muscle-related pathologies in rodents and manncluding hypokalemic period paralysis and muscularysgenesis (Ptacek et al., 1994; Elbaz et al., 1995; Fontainet al., 1994; Jurkat-Rott et al., 1994; Knudson et al., 1989;haudhari, 1992). A different skeletal muscle condition,alignant hypothermia, has in a minority of cases, been

inked to the gene for a2d-1 subunit (Iles et al., 1994).In recent years the first descriptions of CNS-related

isorders linked to VSCC mutations have appeared. Theest characterized of these relate to mutations in the a1A

ubunit gene which induce a range of abnormal statesanging from migraine to ataxia.

utations of the a1A VSCC Subunit Are Associatedith Familial Hemiplegic Migraine

Mutations in the murine a1A VSCC subunit produceoth epileptic and ataxic phenotypes (Fletcher et al.,996; Meisler et al., 1997; Lorenzon et al., 1998; Doyle etl., 1997). Thus far, mutations in the human a1A geneave yet to be linked to any form of epilepsy. Asredicted by the phenotypes of certain mouse mutants

Hess, 1996; Doyle et al., 1997), both spinocerebellar andpisodic ataxias can arise from disruption of the normalunction of the human a1A subunit gene (Riess et al.,997; Jodice et al., 1997; Yue et al., 1997, 1998; Ophoff etl., 1996). In addition, a range of point mutations in theuman a1A gene give rise to familial hemiplegic mi-raine (Ophoff et al., 1996; Terwindt et al., 1998b, 1998a).Familial hemiplegic migraine (FHM) is a relatively

are subtype of migraine with aura that exhibits autoso-al dominant inheritance. Attacks are characterised byigrainous headaches, preceded by or accompanied by

emiparesis and other aura symptoms including speechisturbances, sensory symptoms, and scintillating scoto-as (Whitty, 1986). These symptoms strongly resemble

hose in the much more common condition of migraineith aura (Haan et al., 1994), suggesting that underlyingathophysiological pathways may be shared in these

wo conditions.Linkage studies had indicated that a gene on chromo-

ome 19 p13 was responsible for the inherited defect inbout 50% of families with FHM (Joutel et al., 1993). Oneandidate gene in the correct chromosomal location ishe a1A Ca21 channel subunit. Using association studiesf a number of Dutch families with histories of FHM itas shown that missense mutations in the a1A gene areresent in migraine sufferers of five unrelated families

Table 2; Ophoff et al., 1998, 1996). Analysis of a1A

tructure in a different heritable migraine, familialigraine with vertigo, found no evidence for mutations

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unctional Consequences of FHM Mutations

In the FHM families, the missense mutations all occurn regions of the a1A subunit thought to be important forither voltage-sensing (R192Q) or the conductance prop-rties of the Ca21 selective pore (T666M, V716A, I1811L).he functional consequences of FHM mutations haveeen examined in two different studies, one examiningSCCs containing the human a1A subunit (Hans et al.,999) and the other characterizing mutagenesis of theonserved amino acids in the rabbit a1A orthologue (Kraust al., 1998). As summarized below, both groups describeignificant mutation-induced changes in a1A function.

The R192Q mutation causes an increase in the probabil-ty of opening of a1A channels across a broad range of

embrane potentials. This result is consistent with theosition R192Q close to the voltage sensor in S4 (Hans etl., 1999). Single-channel recording revealed that theutations located in pore lining regions (T666M, V716A,

1811L) all produced changes in the unitary conduc-ance of the channel. In all three mutants, channelpenings to the normal wild-type conductance stateere accompanied by additional openings to a state of

ubstantially lower conductance. This surprising resultndicates that the FHM pore mutations stabilize a

ABLE 2

utations in CACNA1A Coding for Human a1A

Mutation LocationClinical

phenotypeOverallfunction

192Q IS4 FHM Gain666M IIS5-S6 FHM 1 ataxia Loss714A IIS6 FHM Gain

1811L IVS6 FHM 1 ataxia Gaintop codon IIIS1 Ataxia No function (pre

a Thin arrows (Hans et al., 1999); Thick arrows (Kraus et al., 1998).

ABLE 3

ouse Mutants of a1A and Associated Subunits

nimal name Subunit affected Mutation

ottering a1A Missenseeaner a1A New C terminus
ethargic b4 Stop codon, no a binding sitetargazer g2 Early stop codon
hannel conformation that conducts Ca21 less effectivelyhan the major wild-type open channel state. Both studies ofHM mutations also reported effects on the kinetics ofecovery from inactivation (deinactivation). This processas considerably speeded in V716A and I1811L channels

ut was, in contrast, slowed by the T666M mutation (Hanst al., 1999; Kraus et al., 1998). The effects of V716A and1811L are consistent with previous mutagenesis studieshat implicate similar regions of the channel in the process ofnactivation (Hering et al., 1996).

FHM mutations also produce changes in the expres-ion level of a1A-mediated currents (Table 2). Bothncreases (R192Q) and decreases (V716A, T666M, I1811L)n channel density per unit membrane area were re-orted (Hans et al., 1999). This makes it difficult to define

he overall phenotype of the mutations because thectual level of functional channels produced is likely toe specific to the expression system used. Thus expres-ion might be elevated or depressed differentially in differ-nt neurones. At face value, T666M causes loss of function,hile R192Q causes gain of function, while for V716A and

1811L gain of function at the single channel level might beeduced or even reversed by a lower level of expression.

In attempting to consider the functional effects of thearious FHM mutations in vivo we must also bear inind the splice variant background of a1A on which

hey are superimposed. As described above, splicingnd accessory subunit interactions produce consider-

Function: Biophysical detailsa

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ble variation in a1A subunit-mediated currents (Bouri-et et al., 1999; Stea et al., 1994; De Waard and Campbell,995). a1A Channels lacking the valine insertion atosition 421 exhibit significant inactivation (e.g., ‘‘typi-al’’ Q-type currents, see Randall and Tsien, 1995). Foruch channels, the predicted overall effect of the in-reased rate of deinactivation seen in the V716A and1811L mutants is enhancement of total Ca21 influxuring repetitive neuronal activity. In contrast, theehavior of slowly inactivating a1A-V421-mediated cur-ents, such as the ‘‘typical’’ P-type currents of Purkinjeeurones (Regan, 1991; Llinas et al., 1992; Usowicz et al.,992), is likely to be little effected by a change ineinactivation kinetics.Due to the changes in conductance state they produce

as well as a possible decrease in expression), the netffect of the I1811L and V716A mutations are likely to beloss of P-type VSCC function. In contrast, for Q-type

urrents, the negative effects on permeation rate will beounteracted by enhanced deinactivation leading to nohange or even an increase in net channel activity. Itherefore seems that through splicing-induced pheno-ypic variations in a1A containing channels, mutations inhe CACN1A gene are likely to confer different func-ional consequences on different neurones.

Further complexity is potentially introduced by varia-ion in coexpressed accessory subunits. The work of

ans et al. (1999) was performed partly with coexpres-ion of b3a (whole-cell experiments) and partly withoexpression of b2e (isolated patch recordings). The666M mutant whole cell currents were compared foroth b subunits and showed no b subunit dependence.owever, this was the only parameter analyzed so theossibility remains that expression and/or gating of theutants will be sensitive to the accessory subunit

ackground. Kraus et al. (1998) saw similar effects onhole-cell current inactivation with b1a coexpression as

n the human b3a study (Hans et al., 1999). The b4 subunits thought to associate with a1A to produce many of theunctional P/Q type channels in the brain (Liu et al.,996). This subunit combination results in some currentnactivation when expressed with either a1A-a or a1A-b (Bou-inet et al., 1999). It would therefore be interesting toxamine this combination with the FHM a1A subunits.

Other human mutations in a1A and mouse modelsith mutations in accessory subunits produce some-hat more straightforward functional consequences

reviewed by Jen, 1999a). Three nonsense mutations in1A, which produce truncated proteins that are likely toe nonfunctional, all lead to a clinical presentation of
pisodic ataxia. Further, a CAG repeat leading to a C-erminal polyglutamine also leads to a late onset ataxic d
henotype (Zhuchenko et al., 1997). Only one of theseariants (a stop codon in IVS1) produces migraine a inddition to ataxia in some individuals (Jen et al., 1999b).

In addition, two mouse mutants, leaner and totterer,hat show severe ataxia and seizures have missense andplicing errors in a1A, respectively (Fletcher et al., 1996).he mouse models have the powerful advantage that

he functional properties of the native channels can betudied readily. This approach has shown that theseutations lead to a reduction in P-type current density

n cerebellar Purkinje cells (Dove et al., 1998). Anotherouse model, lethargic, exhibits ataxia with seizures.

he mutation is in the b4 subunit and results in aruncated subunit that lacks the a1 subunit interactionomain (Burgess et al., 1999, 1997). This would be predicted

o have a largely similar effect to a b4 knockout, leading to aeduction in Purkinje cell P-type current.

The overall pattern of mutations in a1A suggests thature loss of function mutations lead to a phenotype oftaxia with seizures which are occasionally accompa-ied by migraine (Jen, 1999a). More subtle mutations

hat result in a balance between gain of function in someeurones and loss of function in others seems to confer aigraine phenotype in man. However, even this broad

lassification is blurred by the phenotypes of individu-ls with FHM and ataxia, further emphasising theomplex possibilities for the ultimate effect on a neuro-al network of differential up- and down-regulation of

nhibitory and excitatory neuronal elements.Further experiments with recombinant systems are

equired to fully explore this complexity. Generatingouse transgenics with these human mutations would

e rewarding, as it would enable a functional analysis ofynaptic transmission in situ in brain slices. Studies ofeuronal function in patients will also be highly valu-ble in further elucidating the role of these channelsoth in normal function and in causing the diseasehenotype. This is becoming increasingly achievablesing functional magnetic resonance imaging tech-iques. Ultimately these approaches may provide mucheeded insight into the neuronal mechanisms underly-

ng the initiation of the more common forms of migrainehat affect as many as one in ten adults.

HE MOLECULAR BASIS FORALCIUM-DEPENDENT INACTIVATIONND FACILITATION

odulation of L-Type Channels by Intracellular Ca 21

In addition to the modulation of their properties byifferent accessory subunits, VSCCs are subtly regu-

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ated by a variety of other factors. The most fundamen-al of these, is activity-dependent modulation by intracel-ular calcium ions (Brehm and Eckert, 1978; Ashcroftnd Stanfield, 1982; Imredy and Yue, 1994; Imredy andue, 1992; Yue et al., 1990; Jones and Marks, 1989; Eckertnd Chad, 1984). This form of modulation is mosttriking for the L-type VSCCs and is therefore likely toe important for the fine tuning of the diverse range ofhysiological functions mediated by this family ofhannels (e.g., muscle contraction, control of neuronalene expression and vesicular release from dendrites,ndocrine cells, and ribbon synapses). Within the lastear substantial increases in our understanding of theolecular basis of this form of modulation have beenade. The surprising conclusion from these new studies

s that calmodulin (CaM) acts as both the ion sensor andediator of the feedback effects of Ca21 on VSCC

unction (Peterson et al., 1999; Zuhlke et al., 1999). Evenore of a surprise is the finding that the same molecular

nteraction is responsible for both the positive andegative feedback actions of calcium.Since the work of Brehm and Eckert (1978) it has been

lear that VSCCs operate under intrinsic feedback con-rol. The first such mechanism to be detailed was aegative feedback control produced by Ca21 (Chad et al.,984; Chad and Eckert, 1984), a process that has come toe known as Ca21-dependent inactivation. This impor-ant negative feedback loops is classically observed as aeduction in current flow during maintained depolariza-ion. Elevations of intracellular Ca21 can also enhance-type VSCC activity. In this process, known as facilita-

ion, an increase in current amplitude is observed withppropriate rates of repetitive stimulation (Zygmuntnd Maylie, 1990; Gurney et al., 1989; Noble and Shi-oni, 1981; McDonald et al., 1994). This Ca21-dependent

nhancement of L-type VSCC activity is responsible forhe classical ‘‘staircase effect’’ in cardiac myocytes.

Both the Ca21-dependent inactivation and facilitationf L-type VSCCs are suppressed if Ba21 or Sr21 areubstituted for Ca21 as the permeating ionic speciesBrehm and Eckert, 1978). This points to the involve-

ent of a quite selective Ca21 binding site in theeneration of both Ca21-dependent mechanisms. Thisite has long been suspected to be an integral part of theSCC, most probably the core a1 subunit (de Leon et al.,995; Imredy and Yue, 1994, 1992; Soldatov et al., 1997;uhlke and Reuter, 1998). The most appealing evidence

or this is that even fast binding, high affinity Ca21

uffers such as BAPTA, attenuate but are unable toompletely abolish the feedback actions of permeating
a ions (Neely et al., 1994). This suggests that ‘‘microdo-ains’’ of highly elevated [Ca21] generated close to the
aw

hannel mouth produce either facilitation and/or inacti-ation (Deisseroth et al., 1996).Structure–function experiments on recombinant L-type

SCCs have narrowed the search for the Ca21 bindingite responsible for feedback modulation. Zulke andeuter (1998) demonstrated that the C-terminus regionf the a1C subunit was essential for Ca21-dependent

nactivation. The subsequent identification of an IQaM binding motif within the C-terminal sequence

equired for inactivation then lead to a reexamination ofhe role of this Ca21-binding protein in the processes ofoth inactivation and facilitation.One experimental approach used overexpression of

onfunctional CaM constructs in cell lines also express-ng L type Ca21 channels (Peterson et al., 1999). Thesexperiments showed that a mutant CaM, which wasnable to bind Ca21, could act in a dominant-negativeanner to abolish Ca21-dependent inactivation. This

trongly suggests that CaM is the Ca21 sensor fornactivation and also indicates that endogenous CaM isethered to the channel in a constitutive manner, indepen-ent of Ca21 binding. Parallel biochemical studieshowed that CaM bound to the IQ peptide in the-terminus of a1C with a Ca21-dependence credibly

elated to those demonstrated previously to producenactivation.

In a different study, with recombinant L-typehannels, Zuhlke et al. (1999) explored the effectsf site-directed mutagenesis of the CaM-bindingQ motif. Their manipulations clearly showed that an IQo AQ switch abolished Ca21-dependent inactivation

hile unmasking a process of activity (and Ca21)-ependent facilitation. In contrast, an IQ to EQ switchbolished both Ca21-dependent inactivation and facilita-ion. They also confirmed that the IQ motif region wasble to bind CaM, this data clearly indicates a bifunc-ional role for the CaM–IQ interaction, which presum-bly depends upon the conformational state of the a1

ubunit.

a21-Dependent Modulation of P/Q-Type Channels

Studies of novel protein–protein interactions with the1A subunit have also uncovered CaM-dependent modu-

ation of the P/Q type VSCC. Using the C-terminus ofhe a1A subunit as a bait in a yeast 2-hybrid screen, Lee etl. (1999) pulled out multiple interacting clones thatncoded CaM. Deletion analysis identified a putativeaM binding domain that was confirmed in gel shiftnalysis. Deletion of this 32 amino acid domain from the
1A subunit obliterated a component of inactivation thatas only seen with moderate levels of intracellular Ca21

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uffering (i.e., that afforded by 0.5 mM EGTA). Ca21-ependent facilitation was also abolished in this mutant

Lee et al., 1999). Interestingly, the C-terminal CaMinding domain identified in this yeast 2-hybrid study isot an IQ motif but a sequence with homology to theaM binding domain of type 8 adenylyl cyclase. There

s also an IQ motif in the C terminus of a1A, which isapable of binding CaM (Peterson et al., 1999), a homolo-ous site is additionally located in the a1E subunit. Aset no functional studies of deletions of this second CaMinding domain in a1A or its orthologue in a1E have beeneported.

ethered Calmodulin Is a Common Theme

Historically most data pointed away from, rather thanoward, a role for CaM in Ca21-dependent facilitationnd inactivation of VSCCs. These data, however, can beeconciled with the picture that emerges from the newtudies of Ca21-dependent modulation of recombinantSCCs described above (Imredy and Yue, 1994; Zuhlkend Reuter, 1998; Victor et al., 1997). A key point is thataM appears to be permanently tethered, in a Ca21-

ndependent manner, on or close to the channel. Thisonstitutive anchoring of CaM rather neatly explains aumber of classical experimental observations. These

nclude the failure of classical calmodulin inhibitors likealmidazolium, which require free CaM to be effective,o inhibit inactivation or facilitation (Imredy and Yue,994; Victor et al., 1997; Dasgupta et al., 1989). Tetheringlso explains why Ca21-dependent inactivation persistsn excised membrane patches and lipid bilayers and isesistant to extensive ‘‘washout’’ in whole cell patchlamp recordings (Hofer et al., 1997).

Thus, Ca21-dependent CaM interactions involvinghe C-terminal IQ motif seemingly confer Ca21-depen-ent inactivation and facilitation, at least for the L-typehannels generated by a1C expression. Issues awaitingesolution are the location of the ‘‘tethering site’’ tohich CaM is constitutively bound and the nature of

his seemingly strong interaction, which may or may note on the a1 subunit itself. Wherever CaM is tethered itust be at a site located close enough to the a1 subunit

o effectively sense the microdomain of highly elevateda21 that rapidly forms around the pore mouth follow-

ng channel opening (Deisseroth et al., 1996). In addi-ion, tethering must occur close enough to the IQ site toupport its role in CaM-mediated channel modulation.
n this regard tethering of CaM at the IQ site remains aistinct possibility.
mn

NRESOLVED ISSUES AND FUTUREIRECTIONS

We have described a number of important progres-ions in our understanding of VSCCs that have come toight as we approach the close of this decade. Whiledding to our knowledge, like most significant ad-ances, they bring forth numerous new questions. Thencovering of four new VSCC a1 subunits must featuremong the highlights. This and the isolation of new a2dnd g subunits begs the question of whether there areet more VSCC subunits still hiding in the unchartedeaches of the genome? What is certain is that if novelubunits remain undiscovered they will be found soon,s the sequencing of the entire human genome is comingo completion apace.

The isolation of the genes for three T-type channelsemonstrates a diversity in this family only hinted at byrevious physiological experiments (Huguenard, 1996;ostyuk, 1999). In future years it will be interesting toetermine what specific in vivo roles are played by eachf the three T-type channels. This will doubtless first betudied with targeted gene knockout approaches al-eady underway in a number of laboratories. Subse-uently, it is hoped that the more prolonged process ofrug discovery will lead to subtype selective channelodulators that will both provide additional physiologi-

al information as well as useful therapeutic tools.The role of the now expanded g subunit family is less

lear. The loss of one of these subunits in an epileptichenotype points to an important neurophysiologicalole (Letts et al., 1998), but whether this is truly related toa21 channel function is a matter for debate. Indeed, itas yet to be confirmed that these subunits form trueunctional interactions with other VSCC subunits.

In addition to the ongoing search for novel subunits,ork still remains to be done on the splicing patterns of

nown VSCC subunits and their functional implica-ions. The work described above on the a1A subunit haslready documented parallels for other a1 subunitsncluding the N-type channel encoding subunit, a1B (Lint al., 1997, 1999). Understanding the pharmacology,iophysics, and localization of all possible VSCC spliceariants will be important for building the big picturend may have great implications for developing novelherapeutic agents that target specific VSCC isoforms.

The identification of genetic mutations related toSCC function remains in its infancy. Given the intimate

nvolvement of VSCCs in so many physiological func-ions, the currently identified links to epileptic and

igraine phenotypes will surely be followed up byumerous other disease-related mutations. The identifi-

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ation of such variants will allow the alterations inhannel properties they produce to be carefully charac-erized. Furthermore, through transgenic introductionf mutated channels, better animal models for mutation-

nduced diseases can be generated, thereby speedinghe discovery of suitable therapeutic interventions.

Through the extensive efforts of molecular biologistse are now in possession of a more (and perhaps

omplete) molecular tool kit for the study of VSCCs inoth physiological and pathological processes. Withhis, we can look forward to developing a more thor-ugh understanding of VSCCs on numerous fronts.irected mutational analysis will continue to provide

urther insights in how the molecular structure ofSCCs relates to function; a quest that would be greatly

acilitated by, as yet elusive, high resolution 3D struc-ural data. Our understanding of the growing panoplyf VSCC subunits (and other ion channel targets) willndoubtedly lead to the development of ever moreotent and subunit specific pharmacological agents.long with their potential therapeutic utility, such

ompounds, both alone and combined with gene ma-ipulation methodologies, will allow novel functionalnalysis of VSCC physiology. Data from such studiesill encompass the wide gamut from the generation

nd properties of single submembrane Ca21 microdo-ains, through synaptic transmission in neural net-orks and on to whole animal physiology. The firstecade of the new millennium should prove to be anxciting time for Ca21 channel research.

CKNOWLEDGMENTS

The authors thank Dr. Dave Michalovich for his work in generatinghe VSCC phylogenetic tree. Our thanks also go to Dr. Anna William-on (UCHSC, Denver) and Drs. Orest Hurko and Martin GunthorpeSB, Harlow) for reading and commenting on this manuscript. We arelso grateful to Professor Richard Tsien and his colleagues for access toheir unpublished data on the a1A knockout mouse.

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