Molecular and Functional Properties of the Human alpha 1G Subunit That Forms T-type Calcium Channels

Molecular and Functional Properties of the Human a1G SubunitThat Forms T-type Calcium Channels*

(Received for publication, June 4, 1999, and in revised form, November 10, 1999)

Arnaud Monteil‡§, Jean Chemin‡, Emmanuel Bourinet‡, Gerard Mennessier¶, Philippe Lory‡i,and Joel Nargeot‡

From ‡IGH-CNRS UPR 1142, 141 rue de la Cardonille, F-34396 Montpellier cedex 05, France and ¶CNRS, UMR 5825,Universite de Montpellier II, F-34095 Montpellier cedex 05, France

We describe here several novel properties of the hu-man a1G subunit that forms T-type calcium channels.The partial intron/exon structure of the correspondinggene CACNA1G was defined and several a1G isoformswere identified, especially two isoforms that exhibit adistinct III-IV loop: a1G-a and a1G-b. Northern blot anddot blot analyses indicated that a1G mRNA is predomi-nantly expressed in the brain, especially in thalamus,cerebellum, and substantia nigra. Additional experi-ments have also provided evidence that a1G mRNA isexpressed at a higher level during fetal life in nonneu-ronal tissues (i.e. kidney, heart, and lung). Functionalexpression in HEK 293 cells of a full-length cDNA encod-ing the shortest a1G isoform identified to date, a1G-b,resulted in transient, low threshold activated Ca21 cur-rents with the expected permeability ratio (ISr > ICa >

IBa) and channel conductance (;7 pS). These properties,together with slowly deactivating tail currents, are typ-ical of those of native T-type Ca21 channels. This a1G-related current was inhibited by mibefradil (IC50 5 2 mM)and weakly blocked by Ni21 ions (IC50 5 148 mM) andamiloride (IC50 > 1 mM). We showed that steady stateactivation and inactivation properties of this currentcan generate a “window current” in the range of 265 to255 mV. Using neuronal action potential waveforms, weshow that a1G channels produce a massive and sus-tained Ca21 influx due to their slow deactivation prop-erties. These latter properties would account for thespecificity of Ca21 influx via T-type channels that occursin the range of physiological resting membrane poten-tials, differing considerably from the behavior of otherCa21 channels.

Transmembrane voltage-dependent calcium channels con-trol Ca21 ion entry from the extracellular space and therebyregulate various cellular processes such as muscle contraction,neuronal development and plasticity, secretion, and gene ex-pression. Functional identification of voltage-dependent cal-cium channel subtypes based on their biophysical properties

have distinguished low voltage-activated (LVA)1 and high volt-age-activated (HVA) Ca21 currents. LVA calcium currents,mainly referred to as “T-type currents” due to their fast inac-tivation and small conductance, have been described in a widevariety of cell types (1). Although their physiological functionsstill remain obscure, they are thought to play a role in neuronalburst firing (2), pacemaker activity in heart (3, 4), aldosteronesecretion (5), or fertilization (6). T-type currents are mainlydetected at early stages of development as well as at a giventransition (G1/S) of the cell cycle (7–9). In addition, it has beenreported that enhanced expression of T-type current is associ-ated with a variety of diseases that include cardiac hypertro-phy (10), hypertension (11), and epilepsy (12). Unfortunately,the lack of specific T-type channel blockers has considerablyimpaired their functional characterization. The identificationof the molecular structure of T-type channels has thereforebecome an important challenge in order to unravel their impli-cation in cellular functions (13).

The low level of knowledge about the molecular nature ofT-type channels contrasts with the extensive molecular andfunctional characterization of HVA channels (L-, N-, P/Q-, andR-types) related to the discovery of specific drugs and toxins.HVA calcium channels exhibit an oligomeric structure com-posed of a pore-forming a1 subunit containing voltage sensorsand drug/toxin binding sites, associated with regulatory sub-units (b, a2/d, and g). The functional diversity of HVA calciumchannels is primarily related to the existence of several a1

subunits (a1A–F and a1S) encoded by distinct genes, many ofwhich generate splice variants with specific properties, as de-scribed for the a1A isoforms that generate P/Q-type channels(14).

A search in the genetic data bases for sequences homologous,but not identical, to known Ca21 channel a1 subunits was analternative to conventional molecular cloning strategies for theidentification of novel a1 subunits, and a major advance inT-type Ca21 channel studies has resulted from the identifica-tion of several ESTs and genomic sequences corresponding to asubset of distantly related a1 subunits (15). The full-lengthcDNAs encoding three distinct a1 subunits (a1G, a1H, and a1I)have recently been identified, which give rise, in expressionsystems, to Ca21 channel currents exhibiting hallmarks ofnative T-type currents (15–17). The genes that encode mam-malian a1G, a1H, and a1I proteins are related to the Caenorh-abditis elegans gene c54d2.5 described as a putative Ca21

channel. These recent data (15–18) provide clear evidence that

* This work was supported in part by the Program Genome du CNRS,Association pour la Recherche contre le Cancer Grant ARC9011, andAssociation Francaise contre les myopathies. The costs of publication ofthis article were defrayed in part by the payment of page charges. Thisarticle must therefore be hereby marked “advertisement” in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submittedto the GenBankTM/EBI Data Bank with accession number(s) AF126966(a1G-a) and AF126965 (a1G-b).

§ Supported by Produit Roche (France) and the GRRC (Groupe deReflexion sur la Recherche Cardio-vasculaire).

i To whom correspondence should be addressed. Tel.: 33 499 61 99 36;Fax: 33 499 61 99 01; E-mail: [email protected].

1 The abbreviations used are: LVA, low voltage activated; HVA, highvoltage activated; EST, expressed sequence tag; RACE, rapid amplifi-cation of cDNA ends; aa, amino acid; APW, action potential waveform;PCR, polymerase chain reaction; RT-PCR, reverse transcription-PCR;bp, base pair(s); kb, kilobase pair(s); nt, nucleotide(s).

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 275, No. 9, Issue of March 3, pp. 6090–6100, 2000© 2000 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

This paper is available on line at http://www.jbc.org6090

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genes encoding T-type channels are members of a third sub-family in addition to the gene family encoding neuronal cal-cium channels (a1A, a1B, and a1E) and the gene family encodingL-type calcium channels (a1C, a1D, a1F, and a1S). An importantissue now is to elucidate the molecular basis for the functionaldiversity of T-type Ca21 channels (2).

To address this issue, we have cloned and expressed a hu-man a1G subunit. The molecular data presented here describeseveral new findings concerning the a1G subunit, such as theexistence of several splice variants, the partial gene structure,and the mRNA expression pattern in adult and fetal humantissues. Functional expression was achieved in order to deter-mine the electrophysiological and pharmacological profiles ofthe human a1G-related currents, and we show that the a1G

subunit generates a sustained Ca21 current with specific fea-tures. Characterization of the human a1G subunit is an impor-tant step that significantly extends our knowledge of the mo-lecular and functional properties of the T-type Ca21 channels.

MATERIALS AND METHODS

In Silico Strategies and Probe Design—Genetic data bases weresearched for cDNA and genomic sequences homologous, but not identi-cal, to known a1 subunit cDNAs using (i) sequence similarity search(BLAST; Ref. 19) and (ii) a search for conserved motifs, such as avoltage sensor S4 5 ((R/K)XX)4 (BioMotif).2 Among the various humansequences that were identified, we focused on sequences homologous tothe C. elegans gene c54d2.5 (GenBankTM accession no. U37548). TwoEST clones, H06096 and H19230, available from the IMAGE consor-tium (20), were sequenced and compared with a large set of calciumchannel a1 and sodium channel a cDNAs, using the ClustalW1.7 mul-tialignment software (21). These cDNA sequences were thus defined ascandidates for putative new calcium channel a1 subunits in humansand subsequently used as probes for both Northern blot analysis andscreening of cDNA libraries. The genomic region of chromosome 17q22that contains the CACNA1G gene was recently sequenced (AC004590).The identification of the intron/exon structure was performed using theGRAIL software (22) for the search of putative exons. Alignment of thecDNA sequences characterized in this study, together with the genomicsequence, led us to identify the exons of the CACNA1G gene that forma1G transcripts.

Isolation and Characterization of Human cDNAs—A lgt10 humancerebellum cDNA library (CLONTECH) was screened by conventionalfilter hybridization according to the manufacturer’s protocols, using aprobe generated by PCR using H06096 cDNA as template. This probecovers nucleotides 3660–4590 of the final cDNA. Hybridization wasperformed at 42 °C for 16–20 h in a solution containing 50% formamideusing a random primed [a-32P]dCTP-labeled probe that was added at aconcentration of 5 3 106 cpm/ml. The membranes were then washedwith a final stringency of 0.13 SSC, 0.1% SDS at 65 °C. Three lgt10clones named CC1/CC2/CC3 were identified. An additional EST(H19230) corresponding to the 39 region of the messenger was alsoidentified. The completion of the 59 region of the sequence was achievedusing long reverse transcription-polymerase chain reaction (RT-PCR)protocols (23) on mRNA from human total brain (CLONTECH) usingprimers defined from the rat sequence (Ref. 15; AF027984) and RACE-PCR (24). CC1 and CC3 are overlapping clones that correspond to asplice variation of the human a1G. The resulting isoforms were desig-nated a1G-a (AF126966) and a1G-b (AF126965). The cDNA structure andsequence encoding these subunits was further identified following thecharacterization of two overlapping cDNA fragments of 4056 bp (nt2120 to 3936) and 3432 bp (nt 2840–6272), according to AF126965 (seeFig. 1A). Nucleotide sequences were determined using automatic se-quencing (Applied Biosystems) with the dye terminator strategy. Afull-length cDNA encoding the human a1G isoform designated a1G-b wasconstructed using the unique restriction sites mentioned in Fig. 1A andsubsequently subcloned in the mammalian expression vector pBK-CMV(Stratagene).

Long Range RT-PCR for Native Isoform Identification—A search fornative isoforms of the a1G subunit was further performed using longrange RT-PCR (23) and sequence examination of the cDNA fragmentcovering from nt 2840 (domain II) to nt 6272 (C-terminal region),according to AF126966. The size of this fragment varied from ;3.4 to

;3.8 kb, as a function of the a1G isoform detected. This analysis wasachieved using four different sources of human mRNA: adult wholebrain, adult cerebellum, adult thalamus, and fetal kidney (CLON-TECH). First strand cDNA synthesis (reverse transcription) was per-formed using Superscript 2 (Life Technologies) with a sequence-specificprimer (59-GCTTTCTCCCAACAGCTTC-39) according to the manufac-turer’s protocol. The enzyme was then heat-inactivated (15 min, 70 °C).After cooling on ice, the samples were treated with ribonuclease H (LifeTechnologies, 4 units) for 20 min at 37 °C. PCR amplification wasperformed with 2 ml of the reverse transcription mixture in a finalvolume of 50 ml using the Expand Long Template PCR system (RocheMolecular Biochemicals) with 103 buffer 1 (103 buffer 1: 500 mM

Tris-HCl, pH 9.2, 160 mM (NH4)2SO4, 17.5 mM MgCl2) supplementedwith Me2SO to a final concentration of 5%, 200 nM dNTP, 50 pmol offorward primer (59-CTTCGGCAACTACGTGCTCTTC-39), 50 pmol ofreverse primer (59-TCCTGAAATCCAGCTCAGCTCC-39), and 2.6 unitsof DNA polymerase mix. A hot start PCR procedure was performed withthe following parameters for initial denaturation: 94 °C for 2 min,followed by 30 cycles (10 cycles with denaturation at 94 °C for 10 s,annealing at 65 °C for 30 s, extension at 68 °C for 6 min; and 20 cycleswith denaturation at 94 °C for 10 s, annealing at 65 °C for 30 s, exten-sion at 68 °C for 6 min plus a 20-s increment/cycle) and a final extension(at 68 °C for 7 min). The cDNA fragments were subcloned in pUC18using the Sureclone ligation kit (Amersham Pharmacia Biotech). Sixty-eight positive clones were randomly chosen and fully sequenced usingautomatic sequencing (Applied Biosystems) with the dye terminatortechnology. A similar RT-PCR strategy was used for the identification ofcDNA fragments covering from nt 2120 to 3936 with the forward andreverse primers 59-TAGAGCCCACCAGATGTGCC-39 and 59-GAGAT-GAGCACCAACAGCCC-39, respectively. No evidence for sequence var-iation has been identified to date in this region.

Northern and Dot Blot Analyses—Commercial human Northern blotmembranes (CLONTECH) were hybridized using a 930-bp fragment (nt3660–4590) generated by PCR amplification and random-primed with[a-32P]dCTP. The membranes were treated according to the manufac-turer’s protocol. Relative amounts of mRNA in each line were evaluatedusing two probes: a ubiquitin probe and a b-actin probe as internalcontrols. Two independent dot blot membranes, normalized in theirmRNA abundance by the manufacturer according to the expressionprofile of eight housekeeping genes (Master-blot mRNA membrane;CLONTECH) were treated in similar conditions using a probe coveringthe C-terminal region (nt 6440–6970). The exposure time for Northernand dot blot membranes was 6 days. Densitometric analysis of theautoradiograms was performed using an AlphaImager system (AlphaInnotech Corp.) in order to provide semiquantitation of a1G mRNA ineach condition. No signal was observed for internal DNA controls,except for human genomic DNA.

Transient Transfection—A plasmid encoding the reporter gene CD8(25) was used in transient transfection experiments, together with thepBK-CMV construct that encodes for the a1G-b subunit, in a 1:10 ratio.Human embryonic kidney cells HEK-293 were grown in Dulbecco’smodified Eagle’s medium (Eurobio) supplemented with 10% fetal bovineserum and 1% penicillin/streptomycin (v/v). For optimal transfection,cells were plated at 30–40% confluence on Petri dishes coated withpoly-L-ornithine (Sigma). A standard calcium phosphate transfectionprocedure was performed. Positively transfected cells were identifiedwith anti-CD8 antibody-coated beads (Dynal) and further analyzed byvoltage clamp experiments. cDNAs encoding the rat brain a1A-a, a2/d1b,and b1b subunits were inserted in the vertebrate expression vectorpMT2 (14) and cotransfected in HEK 293 cells as a mix of a1A-a, a2/d1b,b1b, and CD8 cDNAs at a molar ratio of 1:1:1:0.1.

Electrophysiology—Macroscopic currents were recorded by thewhole-cell patch clamp technique at room temperature (;21 °C) usingan Axopatch 200B amplifier. Data were acquired on a PC computerusing the pClAMP6 software suite (Axon Instruments). Records werefiltered at 5 kHz. Leak and capacitive currents were subtracted using aP/25 procedure when needed (i.e. for tail current recordings). Extracel-lular solution contained 2 mM CaCl2 (or 2 mM BaCl2 or 2 mM SrCl2), 160mM TEACl, 10 mM HEPES (pH to 7.4 with tetraethylammonium hy-droxide). Pipettes made of borosilicate glass with a typical resistance of1–2 megaohms were filled with an internal solution containing 110 mM

CsCl, 3 mM MgCl2, 10 mM EGTA, 10 mM HEPES, 3 mM Mg-ATP, 0.6 mM

GTP (pH to 7.2 with CsOH). For pharmacological experiments, drugswere prepared daily in the external medium, freshly (amiloride andmibefradil) or from a stock solution (NiCl2). The various dilutions wereapplied to cells by a gravity-driven homemade perfusion device, con-trolled by solenoid valves. Single channel recordings were done in thecell-attached mode. Sylgard coated pipettes (resistance of 7–15 mega-2 G. Mennessier, submitted for publication.

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ohms) were filled with a solution containing 110 mM BaCl2, 10 mM

HEPES (pH to 7.2 with TEAOH). Membrane potential was reducedtoward 0 mV by bathing the cells in a high potassium solution contain-ing 140 mM potassium gluconate, 10 mM EGTA, 10 mM glucose, 1 mM

MgCl2, 10 mM HEPES (pH to 7.3 with KOH). The sampling frequencyfor acquisition was 10 kHz, and data were filtered at 1 kHz.

Data were analyzed using pCLAMP6 (Axon Instruments), Excel (Mi-crosoft), and GraphPad Prism (GraphPad Inc.) software programs.Whole cell current-voltage curves were fitted using a modified Boltz-mann equation as described previously (26). Activation and inactivationcurves were fitted with a Boltzmann equation. Apparent dissociationconstants (Kd) were obtained from the fitting of the drug dose-responsecurves using a sigmoidal function. Results are presented as the mean 6S.E., where n is the number of cells used.

RESULTS

Cloning of a Human a1G Subunit—Several cDNA clonescovering from domain II to the C-terminal region of the humana1G isoform were isolated from a cerebellum cDNA libraryusing a probe designed from the identified EST H06096 (Fig.1A). Additional cDNA clones that cover the entire coding regionof the human a1G were identified using RT-PCR and RACE-PCR from human total brain mRNA. Finally, two overlappingcDNA fragments of 4 and 3.4 kb covering from nt 2120 to 6272were obtained by RT-PCR from human total brain mRNA tofurther confirm the cDNA structure of human a1G isoforms. Afull-length cDNA encoding the a1G-b isoform was then con-structed using the overlapping cDNAs (Fig. 1A).

Alignment of our cDNA sequences to the correspondinggenomic region of chromosome 17q22 (AC004590), togetherwith the use of GRAIL software (22) for the identification ofputative exons in the genomic sequence, led us to identify thepartial intron/exon structure of the CACNA1G gene that en-codes the a1G isoforms (Fig. 1B). The CACNA1G gene covers

,70 kb in the 17q22 region. A total of 41 putative exons waspredicted using GRAIL software (Fig. 1B). The 59-end ofCACNA1G (;500 bp), which has been determined recently(27), is comprised within a single exon. Accordingly, this exonthat also contains the first ATG codon was designated exon 1.This ATG is comprised within a consensus site for initiation oftranslation: AXXATGG (28). The coding region of the two hu-man a1G subunits described later in this study is composed of34 exons. One of these two variants which has the highesthomology with the original rat sequence (15), was named a1G-a.The isoform a1G-b resulted from the use of an alternate 59-splicedonor site of exon 25 combined with the acceptor site on exon 27(Fig. 2, A and B). Four additional exons were identified inRT-PCR experiments conducted on mRNAs from human totalbrain, cerebellum, thalamus, and fetal kidney. These exonsencode insertions within the II-III loop (insertion e), within theIII-IV loop (insertion c), and within the COOH-terminal region(insertions f and d) (Fig. 2A). In our experiments, the insertionc, which corresponded to the use of exon 26 (Fig. 2B), was foundonly in combination with variation b. Three other predictedexons that are included in Fig. 1B have not been retrievedexperimentally to date. Sequencing of cDNA fragments ob-tained using long range RT-PCR experiments was performed inorder to determine the potential association of the a/b, c, d, e,and f regions in a1G transcripts as well as their relative abun-dance in native human tissues. A total of 68 cDNA fragmentsobtained from human total brain, cerebellum, thalamus, andfetal kidney mRNAs was analyzed (Fig. 2C). A majority of a1G-a

isoform alone, or in combination with insertion e, was found inneuronal tissues. By contrast, only the a1G-b isoform, mostlyassociated with insertion c, was detected in fetal kidney.

FIG. 1. Cloning and molecular properties of human a1G cDNAs. A, the cloning strategy presented under “Materials and Methods.” Severalpartial cDNA fragments were isolated from lgt10 human libraries (CC1, CC2, and CC3). Two ESTs and two PCR fragments covered the 59- and39-ends. Two additional PCR fragments of 4 and 3.4 kb, according to a1G-b sequence (AF126965), were subcloned and sequenced. This latter PCRfragment was used further for a1G isoform determination as described under “Materials and Methods” and in the legend to Fig. 2. A full-lengthcDNA was constructed using overlapping PCR strategies as well as the unique restriction sites. An additional restriction site (BssHII) wasintroduced by PCR in the cDNA covering the untranslated 59 region to allow final assembly and subcloning in the pBK-CMV vector. B, the partialintron/exon structure of the human gene CACNA1G was determined from sequence AC004590 using the GRAIL software for exon prediction.Exons are boxed on the bottom line. Black boxes correspond to the 34 exons that are used in the human isoforms a1G-a and a1G-b. The ATG and TGAcodons are comprised in exon 1 and exon 38, respectively. Œ, exon 25 that is alternatively spliced to produce the two isoforms a and b. *, exons 14,26, 34, and 35 that were identified in RT-PCR experiments. l, three predicted exons that are not numbered, since they have not been detectedin RT-PCR experiments.

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The construct a1G-a encodes a 2250-amino acid (aa) proteinwith a calculated molecular mass of 249.333 Da. Primary se-quences of the two variants a1G-a and a1G-b were compared withthe rat and mouse a1G proteins (Fig. 3). The four transmem-brane domains are highly conserved (98–100% identity). Theconnecting loops between domains I and II and between do-mains II and III, as well as the NH2 and COOH-terminalregions are more divergent (85–95% identity). Within the II-IIIloop, overall identity is 90% without taking into account inser-tion e (23 aa), which occurs in human, rat, and mouse (Fig. 3A).The two variants a1G-a and a1G-b encode a distinct intracellularIII-IV loop (Fig. 3B). By contrast with the mouse a1G sequence,insertion c (18 aa) was found associated only with the splicevariant b in our experiments (Fig. 2C). Nevertheless, this in-tracellular loop is shorter (50–70 aa) compared with others(;300 aa). The III-IV loop in isoform a1G-a is 100% identical tothe rat a1G sequence. The other isoform, a1G-b, exhibits ashorter III-IV linker resulting from a 21-bp deletion describedabove. Sequence comparison of the III-IV loop of several a1G,a1H, and a1I subunits, as well as the putative c54d2 geneproduct, revealed that sequence variability within this intra-cellular loop is highly related to the length of this region. The

sequences surrounding this insertion are more homologous,and several aa are conserved (Fig. 3B). The aa sequences cor-responding to insertions f (48 aa) and d (45 aa) in the C-terminal region of the human a1G subunit that are presented inFig. 3C have not been described to date. The search for putativeregulatory sites, such as consensus phosphorylation sites forprotein kinase A and protein kinase C, led us to identify eightprotein kinase A and up to 20 protein kinase C sites containedwithin the intracellular regions. Most of these consensus sitesare conserved among human and rat sequences. Interestingly,a protein kinase A/protein kinase C consensus site identified inloop III-IV for a1G-a is removed in the isoform a1G-b, as aconsequence of splicing of exon 25 (Fig. 3B).

Distribution of a1G mRNA in Human Tissues—Northern blotanalysis of a large variety of adult human tissues showed thata1G mRNA is predominantly present in the brain (Fig. 4A).Nonneuronal tissues that were positive were ovary and pla-centa. A very weak band was also observed in testis, smallintestine, colon, and heart. Positive tissues exhibited a majorband of approximately 8.5 kb along with discrete larger bands.The reason for a smaller band in the prostate mRNA lane iscurrently unknown. Considering neuronal tissues, cerebellumas well as thalamus and substantia nigra displayed a strongsignal (Fig. 4B). Several other tissues from the central nervoussystem also revealed a significant level of a1G expression, whileother areas, like hippocampus, showed a weaker a1G mRNAsignal. The pattern of a1G mRNA expression identified inNorthern blots was mostly confirmed using dot blot analysiswith densitometric quantification (Fig. 5). Two independent dotblot membranes were examined (see “Materials and Methods”).Brain tissues showed the strongest signal (thalamus . occipi-tal lobe . cerebellum). The only difference between dot blot andNorthern blot analyses concerns the spinal cord, since expres-sion of a1G mRNA was undetectable on Northern blot, althoughthe use of two independent internal controls, ubiquitin andb-actin, suggested that the quality this mRNA sample wascorrect on the Northern blot membrane. Altogether, dot blotexperiments revealed a level of a1G expression in good agree-ment with Northern blot analysis for other neuronal tissues.

It is worth underlining the fact that analysis of two inde-pendent dot blot membranes revealed that the a1G mRNAexpression level was significantly higher in fetal peripheraltissues, heart, kidney, lung, spleen, and thymus, comparedwith adult tissues (Fig. 5B). This pattern of expression was notdisplayed in the brain, since the amount of a1G mRNA wasequivalent in adult and fetal brain. Since dot blot membranesare normalized for the amount of mRNA present in each dot(see “Materials and Methods”), we have performed a densito-metric analysis that shows the relative abundance of a1G

mRNA in these tissues (Fig. 5B). Altogether, these data indi-cate that the expression of a1G is developmentally regulated.

Electrophysiological Description of the Human a1G Cur-rents—Functional properties of human a1G-dependent chan-nels were investigated using the a1G-b isoform, as expressed inHEK-293 cells. This a1G-b isoform corresponds to the minimalstructure identified to date for the native human a1G subunit.Robust T-type Ca21 currents were recorded in the presence of2 mM Ca21 (Fig. 6A). Activation kinetics was voltage-depend-ent, with time constants ranging from 10 to 1.2 ms for poten-tials between 250 and 130 mV. Inactivation kinetics was alsostrongly voltage-dependent. Current decay was best describedby a single exponential function, with time constants rangingfrom 35 to 12 ms between 250 and 130 mV. This propertyimplied criss-crossing of current traces as observed for nativeT-type currents (29).

This current activated around 260 mV and peaked at 230

FIG. 2. Identification of a variety of isoforms for the humana1G subunit. A, schematic structure of the a1G subunit that illustratesthe localization of the a/b variation generated by alternate splicing ofexon 25, as well as the positions of the insertions e, c, f, and d encodedby exons 14, 26, 34, and 35, respectively. B, description of the alterna-tive splicing mechanisms that are expected to give rise to the a1G-a,a1G-b, and a1G-bc isoforms. Summary of the analysis of the PCR-derivedcDNA fragments encoding the 3.4–3.8-kb fragment (from domain II toC terminus). A total of 68 clones deriving from total brain (22), cerebel-lum (22), thalamus (7), and fetal kidney (17) were analyzed by sequenc-ing, and the occurrence of each combination is reported. Note that atruncated form of a1G (11*) was found three times. This truncation (102bp) removes most of the IIIS6 region of the protein.



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FIG. 3. Multiple alignments and primary sequence analysis of the human a1G subunit. A, multiple alignments performed withClustalW1.7 (blosum matrix) of the deduced aa sequences that encode for the intracellular loop between domain II and domain III of a1G. The



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mV, while reversal potential was observed at 150 mV (Fig. 6B).When calcium (Ca21) was replaced with equimolar barium(Ba21) or strontium (Sr21) ions, a1G currents reflected perme-ation properties typical of that of T-type calcium channels (Fig.6C). The amplitude of Ba21 currents was smaller (permeabilityratio of Ba21/Ca21 ;0.96), while Sr21 currents were larger(permeability ratio of Sr21/Ca21 ;1.35) than Ca21 currents(n 5 6; Fig. 6D). Interestingly, Ba21 current inactivation ki-netics was faster, compared with Ca21 current. Single channelcurrents were measured in the presence of 110 mM Ba21 (Fig.6E), using tail current protocols (16). Unitary current ampli-tude was recorded for membrane potentials ranging from 2120to 0 mV, indicating a slope conductance of 7.3 pS (n 5 3; Fig.6F). Current amplitude at 0 mV was 20.39 6 0.2 pA (n 5 3).

Pharmacological Properties of Human a1G Subunit—Wehave studied the sensitivity of human a1G channels to threemolecules, nickel (Ni21), amiloride, and mibefradil, that areconsidered to be T-type channel blockers. The divalent ion Ni21

was described as a potent blocker of T-type currents; however,block of the Ca21 currents generated by the human a1G subunitwas modest, with an IC50 of 148 6 10 mM (n 5 8; Fig. 7A). Thisa1G current was also poorly sensitive to amiloride, since lessthan 30% of inhibition in the current amplitude (n 5 5) wasobtained in the presence of 1 mM amiloride (not shown). Fi-

nally, mibefradil, considered as the most potent T-type channelblocker (30), affected a1G current amplitude with an IC50 of 2mM (n 5 7; Fig. 7B) in good agreement with previous studies onnative and recombinant T-type channels.

FIG. 4. Northern blot analysis of human a1G mRNAs from avariety of tissues with commercial membranes (CLONTECH)using the same probe as for the human cerebellum cDNA libraryscreening. Internal control was performed using a ubiquitin (Ubiq.)and a b-actin (not shown) probe provided by the manufacturer (CLON-TECH). A, the pattern shows strong expression in brain but also inheart, placenta, and small intestine. B, the expression profile for avariety of human central nervous system regions is shown.

FIG. 5. Dot blot analysis of adult and fetal tissues. Dot blotanalysis of human a1G transcripts with commercial membranes(CLONTECH) using a probe that covers the 39-end of the transcript(exon 38, nt 6440–6970) is shown. A, relative intensities of the spotswere determined using the AlphaImager system (Alpha InnotechCorp.). B, comparison and quantitation of a1G transcripts in fetal (blackbar) and adult (white bar) tissues were determined by the densitometricanalysis on two independent master blot membranes (designated dot 1and dot 2).

sequences presented are human a1G-a (ha1Ga; AF126966); human a1G-b (ha1Gb; AF126965); human partial cDNA NBR13/a1G-c (ha1G; AB012043),which carries out insertion e (1e); rat a1G isoform 1 (ra1G1 (15); AF027984); rat a1G isoform 2 (ra1G2; AF125161); mouse a1G (mA1G (30);AJ0112569). One should note that the 23-aa insertion is retrieved in human, rat, and mouse. #, insertion; *, nonconserved aa substitution; twodots:, strongly similar aa substitution; one dot, weakly similar aa substitution. B, multiple alignments performed with ClustalW1.7 of the deducedaa sequences that encode the intracellular loop between domain III and domain IV of a1G. In addition to the set of sequences defined above, thesequences encoding for the C. elegans c54d2 (U37548); human a1H isoform 1 (ha1H1 (16); AF051946); human a1H isoform 2 (ha1H2 (18);AF073931); and rat a1I (ha1I (17); AF086827) are added. Symbols are as defined for A. The symbols in the top line show the diversity of c54d2 withthe mammalian sequences. The bottom line illustrates the aa diversity among the various mammalian a1 subunits encoding T-type channels. Notethat a putative protein kinase A/protein kinase C phosphorylation site (#) is removed in the human isoform a1G-b (AF126965; ha1Gb). C, alignmentof the C-terminal region of several human a1G isoforms that carry out no insertion (2d2f; AF126966), insertion f (1f), or insertion d (1d), togetherwith the mouse and rat sequences.



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Gating Properties of Human a1G Channels—The voltage de-pendence of the channel activation was determined by meas-uring tail current amplitudes at various depolarizing pulses(Fig. 8, A and C) that fully activates a1G currents. For thatpurpose, duration of each pulse was adjusted to the averagetime-to-peak current values presented in the upper part of Fig.8A. Normalized amplitudes of tail currents plotted as a func-tion of the membrane potential described a biphasic smoothcurve for steady state activation, which could be best fitted bythe sum of two Boltzmann functions (Fig. 8C) with half activa-tion values of V1 5 241.8 mV and V2 5 214.7 V (n 5 5). Steadystate inactivation properties were determined using a standarddouble pulse protocol (Fig. 8B). A 5-s prepulse (2100 to 235mV) preceded a test pulse at 230 mV. Data were fitted by asingle Boltzmann function with V0.5 5 271.6 mV (Fig. 8C).Superimposition of the steady state activation and inactivationcurves revealed the existence of a window current in the range

of 265 to 255 mV (Fig. 8C).Deactivation Properties of a1G-related Channels—The deac-

tivation properties of human a1G channels were deduced fromtail current analysis (Fig. 9). Fig. 9A shows that kinetics of tailcurrents was slower when repolarizing membrane potentialswere more positive, reflecting a strong voltage dependence ofthe deactivation process for a1G-related currents (Fig. 9B).Additional experiments using action potential waveforms(APWs) as a voltage clamp command were designed to evaluatewhether a1G-related channels allow brief or sustained entry ofCa21 in the range of physiological membrane resting potentials(about 275 mV). When HEK cells overexpressing the humana1G subunit were stimulated with neuronal APWs (2-ms dura-tion) applied from a holding potential of 275 mV, a slow inwardCa21 current following the APW was recorded (Fig. 9C). Thiscurrent was totally suppressed by the application of 1 mM

mibefradil (not shown). By contrast, application of this APWprotocol to cells that expressed HVA current generated by a1A

subunit resulted in a transient inward calcium current thatcorrelates with the duration of the APW (Fig. 9C). A similarresult was obtained using native neurons expressing only HVAcurrents (not shown).

DISCUSSION

This study is the first description of the molecular and thefunctional properties of a cloned a1G T-type Ca21 channel sub-unit from humans. Specific findings include (i) the description

FIG. 6. Electrophysiological properties of the currents gener-ated by the human a1G subunit. A, currents evoked by increasingdepolarizations from 2100 mV to 150 mV with 10-mV increments(from holding potential of 2100 mV). The arrow indicates the peakcurrent recorded for a test pulse to 230 mV. Note the criss-crossingpattern of current traces. B, averaged current density/voltage relation-ship obtained from 15 cells. C, illustration of the permeation profile ofa1G channel for divalent cations. Equimolar substitution from Ca21 toBa21 ions induces a slight decrease (5%) in peak current amplitude,while equimolar substitution with Sr21 increases current amplitude(35%). D, mean Ba/Ca and Sr/Ca permeability ratios obtained frompeak current amplitude values measured in 2 mM Ca21, Ba21, and Sr21

(n 5 6). E, single channel measurements of a1G currents recorded usinga tail current protocol (see Refs. 16 and 17). The voltage protocolincludes a 10-ms step to 130 mV followed by a test pulse to theindicated potentials. F, unitary current amplitudes were obtained atvarious potentials (2120 to 0 mV) and plotted as a function of the testpotential value. Linear regression revealed a slope conductance of 7.3picosiemens (n 5 3).

FIG. 7. Pharmacological profile of a1G channels. A, mean dose-effect curve for Ni21 (n 5 8) shows an IC50 of 148 6 10 mM. The effect ofincreasing Ni21 ion concentrations on an a1G current on peak Ca21

current amplitude is shown in the inset. The current was evoked by a50-ms depolarization at 230 mV (stimulation rate of 0.1 Hz). Currenttraces from bottom to top correspond (in mM Ni21) to control, 1, 10, 30,100, 300, and 1000. B, the mean dose-effect curve for mibefradil indi-cates an IC50 of 2 6 0.2 mM (n 5 7). The inset illustrates the effect ofincreasing concentrations of mibefradil on a1G current evoked using thesame protocol as in A. Current traces from bottom to top correspond (inmM mibefradil) to control, 0.1, 0.3, 1, 3, and 10.



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of several a1G isoforms generated by alternative splicing, (ii)evidence for a developmental regulation of a1G transcripts, and(iii) the description of specific functional properties related toslow deactivation and the existence of a window current in therange of the resting membrane potential.

Our study provides the first evidence for alternatively

spliced isoforms of the a1G subunit, revealing therefore that themolecular diversity of T-type channels not only relies on theexpression of three subunits encoded by distinct genes, a1G,a1H, and a1I (17). In humans, the gene CACNA1G encoding a1G

is localized on chromosome 17q22 (15). Exon prediction usingGRAIL (22) led us to identify 41 putative exons within thegenomic sequence that covers the coding region of that gene(AC004590). We have numbered 38 of them that were unam-biguously identified in cDNA cloning and RT-PCR experi-ments. Whether additional exons exist cannot be excluded.Recently, Toyota et al. (27) have performed 59-RACE analysis ofCACNA1G in order to identify the transcription start site ofthis gene in humans (GenBankTM accession no. AF124351). Ingood agreement with our results, no additional exon was foundupstream to the one identified here as exon 1. More impor-tantly, these authors have demonstrated that the CACNA1Ggene is found inactivated by aberrant methylation in severalhuman tumors.

We have identified here that the a1G-b isoform corresponds to

FIG. 8. Steady state activation and inactivation of a1G calciumcurrent. A, determination of the steady state activation was based onthe measurement of tail current amplitudes according to the protocolpresented in the upper part of A. For each depolarization, the tailcurrent amplitude was measured for pulse durations adjusted from themembrane potential/time-to-peak plot shown in upper part of the figure.B, steady state inactivation was estimated from the measurement of thecurrent amplitude at 230 mV after a 5-s predepolarization pulse ofincreasing amplitude (2100 to 235 mV, 5-mV increment). C, normal-ized steady state activation obtained from the protocol described in A(open symbols, n 5 5). The smooth curve corresponds to the best fitobtained using two Boltzmann functions with normalized activation(m`) being a function of the test potential (V) according to the equationm` 5 A1/1 1 exp((V1 2 V)/K1) 1 A2/1 1 exp((V2 2 V)/K2), where A1 andA2 are the respective contributions of each Boltzmann distribution; V1and V2 are the two half-activation values; and K1 and K2 are the slopes.Values from the fit are A1 5 57%, A2 5 43%, V1 5 241.8 mV, V2 5 214.7mV, K1 5 5.1 mV, and K2 5 11.9 mV. Mean steady state inactivationcurve (filled symbols, n 5 10) was best fitted with a single Boltzmanndistribution with a half-inactivation potential (V0.5) of 271.6 mV and aslope of 4.33 mV. Superimposition of steady state activation (m`) andinactivation (h`) curves revealed a window current near 265 to 255mV.

FIG. 9. Deactivation properties of a1G currents and APW volt-age clamp experiments. A, a1G currents were activated by a 7-ms testpulse at 230 mV from a holding potential of 2100, and a family of tailcurrents was recorded for subsequent repolarizations from 2120 to 240mV. B, plot of mean deactivation time constants as a function of mem-brane repolarization potential (n 5 5). C, Ca21 currents generated bythe a1G and a1A subunits (see labels) in response to a neuronal APW(2-ms duration; holding potential of 275 mV). Note that, according tothe slow deactivation kinetics of the currents generated by the a1Gsubunit, activation and peak of the Ca21 current occurred during repo-larization. This inward current persisted for more than 10 ms afterrepolarization. In contrast, HVA current generated by the a1A-a subunitusing the same APW protocol evoked a transient Ca21 current thatmatches the APW duration.



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the minimal structure of the native a1G subunit in humans,and therefore it is important to consider that analysis of thisisoform provides a basis for the comparison of the deduced aasequences of a1G subunits among species, as well as the iden-tification of regions that are critical to explore for furtherstructure-function studies. Indeed, a mouse a1G subunit re-cently described (31) exhibits two insertions within loops II-IIIand III-IV, compared with rat a1G (15). We demonstrate herethat the insertion in loop II-III corresponds to the use of anadditional exon (exon 14 as referred to in the human sequence).We have identified the expression of exon 14, i.e. insertion e,occurs in human brain mostly in association with region a. Inaddition, we have identified two novel insertions d and f in theC-terminal region of the human a1G subunit. The occurrence ofthese latter insertions is low: 1 and 7 out of 68 for d and f,respectively. The functional role of these insertions in II-IIIloop and the C terminus remain to be determined. Unexpect-edly, while sequencing our batch of 3.4/3.8-kb fragments thatcovered from domain II to the C-terminal region, we have founda truncated form of a1G cDNA (3 out of 68) that corresponds toa 102-bp deletion (nt 4498–4601; AF126965) of a region thatencodes the IIIS6 segment. The reason for the existence of sucha truncated form of a1G, which corresponds to an aberrantalternative splicing of exon 25, is unclear at the present time.

The differences among human, mouse, and rat a1G subunitswithin the III-IV loop are likely to correspond to the combina-tion of two mechanisms. First, we have demonstrated here thatthe use of an alternate splice site within exon 25 can generatethe two human a1G isoforms, a1G-a and a1G-b. We referred hereto these isoforms as a1G-a and a1G-b, by comparison to theoriginal rat a1G subunit (AF027984), which is identical to thehuman a1G-a. A similar mechanism is likely to occur in rodents,since a sequence similar to the III-IV loop of a1G-b can beretrieved in rat (32). Second, human, mouse, and rat exhibit anadditional insertion, designated c, in this region (31, 32). In ourstudy, insertion c was found only in combination with b, result-ing in a1G-bc isoforms that were mostly expressed in fetal kid-ney. Again, the role of insertion c in the human a1G subunit hasyet to be determined. Altogether, we describe here two mech-anisms that are able to generate a1G isoforms with a distinctIII-IV loop. The isolation and tissue distribution of cDNAsencoding the various human a1G isoforms as well the investi-gation of their functional properties is now an important issue,as demonstrated for the a1A isoforms that give rise to P/Q Ca21

channel subtypes (14).A common feature of the human and rodent a1G subunits, as

well as for the a1H and a1I subunits, is the absence of theb-subunit binding site, called AID (33), found in every HVA a1

subtype. Such data are consistent with previous experimentsshowing that b-subunit knock-down using antisense strategiesdoes not affect T-type currents (34, 35). Similarly, the G proteinbg binding sites described in the non-L a1 subunits (36) are notretrieved. This is also the case for the Ca21 binding and cal-modulin binding domains involved in calcium dependent inac-tivation (37, 38). Several putative phosphorylation sites forprotein kinase A, protein kinase C, and CaM-kinase II occur inthe sequence, but their functional relevance needs to be inves-tigated. Interestingly, the shortest isoform, a1G-b, lacks a pu-tative protein kinase A/protein kinase C phosphorylation sitein the III-IV loop.

Based on the a1G-a isoform, the aa sequence of the III-IV loopis highly conserved among the three subunits a1G, a1H, and a1I,which would suggest a conservation in function (75%; Ref. 17).It was proposed that this connecting loop might be importantfor inactivation of T-type channels (17, 39) on the basis of thatdescribed for Na1 channels (40). However, although the vari-

ous a1G isoforms exhibit important sequence variations in theIII-IV loop, inactivation properties of currents related to thehuman a1G-b subunit did not show significant differences fromthose of currents related to rodents a1G isoforms (15, 31). Fur-ther work is needed to clearly examine whether this region ofa1G influences the voltage-dependent inactivation process and,subsequently, if sequence variations within the III-IV loophave functional consequences on T-type Ca21 channel activity.

Northern blot and dot blot analyses have clearly indicatedthat the highest level of a1G expression occurs in human braintissues, particularly in thalamus, cerebellum, substantia nigra,and frontal and occipital lobes. Overall, these data correlatewell with those of the recent study by Talley et al. (41). Expres-sion of a1G in thalamus is expected to underlie the low thresh-old spike activity described in thalamic neurons (42). Further-more, overexpression of T-channels in this structure has beenobserved in GAERS rats, an animal model of absence epilepsy(12). More importantly, our data have revealed that the a1G

subunit expression is developmentally regulated. While a1G israther similarly expressed in adult and fetal brain, it is muchmore predominant in fetal peripheral tissues such as heart,kidney, or lung. Enhanced expression of an a1 subunit encodingT-type channels in fetal heart correlates well with electrophysi-ological data, since it is generally accepted that T-type currentsare preferentially recorded in embryonic or newborn myocytes(43). It complicates, however, the identification of the so-calledcardiac T-type channel, since another member of the T-typechannel family, a1H, has been cloned from heart tissue (16).The relative expression of both a1H- and a1G-related channelsin cardiovascular tissues is therefore an important issue thatneeds to be further investigated.

The electrophysiological and pharmacological properties ofhuman a1G currents were examined in a physiological externalcalcium concentration (2 mM), except for the determination ofthe single channel conductance (;7 picosiemens). Overall, bio-physical properties such as low activation threshold (,260mV), potential for peak current (230 mV), activation and inac-tivation kinetics, cross-over of current traces, and permeationproperties (ISr . ICa $ IBa) are typical of that of T-type currentsrecorded in native cells. Our data are also in good agreementwith those of other cloned a1G channels, although comparisonis impaired by the differences in experimental conditions. Forinstance, threshold of activation at more positive potentials(250 mV) and a shift of the I/V curve (peak at 210 mV)reported for the mouse isoform (30) is likely to be due to the useof high concentrations (20 mM) of divalent cations. It is worthnoting that steady state activation is best described by the sumof two Boltzmann distributions. Similar results were obtainedfor human a1H channels (18) and rat a1G channels (44). Akinetic model that includes several closed state transitionsprior to opening was proposed in Ref. 44, and this scheme canaccurately account for most of the qualitative and quantitativefeatures of human a1G channels described here. By contrast, asingle Boltzmann function fits the steady state inactivation.This dual behavior for activation and inactivation is reminis-cent of that reported for several other channel types, includingN-type in dorsal root ganglion neurons (45), for which a bimo-dal activation process (i.e. “willing”/“reluctant” modes) was cou-pled to only one inactivation process.

The pharmacological profile of human a1G-related channelswas determined using the few molecules, nickel (Ni21), mibe-fradil, and amiloride, which have been used to discriminateLVA T-type calcium channels from HVA calcium channels. Thehuman a1G channel has a relatively high sensitivity to mibe-fradil with an IC50 in the micromolar range. This value is quitecomparable with the mibefradil inhibition of human a1H cur-



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rents (1 mM) recorded in the presence of 15 mM Ba21 (18).Amiloride seems to affect differentially a1G- (IC50 . 1 mM) anda1H-related currents (IC50 5 167 mM; Ref. 18), although thedifferences in the recording conditions should again be consid-ered. Similarly, we found a rather low sensitivity to Ni21 ionsof human a1G currents (IC50 5 150 mM), as compared with a1H

calcium channels for which an IC50 of 6 mM was reported (18).One should note that the mouse a1G channel is blocked by aneven higher Ni21 concentration (IC50 . 1.1 mM in 20 mM Ca21;Ref. 31). Nevertheless, discrepancies in the sensitivity to Ni21

ions are consistent with the data reported on native cells (2). Itis important to note that native T-type channels from brainregions expressing a high level of a1G subunit exhibit lowsensitivity for Ni21 ions in good agreement with that describedfor expressed a1G channels (2, 41, 46). A consequence of thesedata is that the block by micromolar concentrations of Ni21

should not longer be considered as distinguishing LVA fromHVA channels (47).

A hallmark of T-type channels is the slow kinetics of theircurrent deactivation, and our data clearly show that the deac-tivation kinetics of the Ca21 current related to human a1G

channels is voltage-dependent. We further demonstrate thatthe consequence of a slow deactivation process can be visual-ized using neuronal APWs as a voltage clamp command. Over-expression of T-type calcium channel in HEK cells greatlyimproves such investigations, and we show that APW stimula-tion (,2 ms) from cell’s resting potential values (275 mV)induces a sustained Ca21 current that occurs during repolar-ization and persists for more than 15 ms. The kinetics of thisCa21 current is consistent with the kinetics of tail currentsevoked by a square-pulse with membrane repolarization at thispotential. As expected, this Ca21 influx is likely to be shaped bythe pattern of APWs, such as duration, resting membranepotential, and firing rate (48). Deactivation is a feature thatdistinguishes LVA/T-type currents (t ; 5 ms; Ref. 2) from HVAcurrents (t ; 0.3 ms; Ref. 49). McCobb and Beam (50) havereported that distinct Ca21 signals are generated by LVA andHVA channels in voltage clamp experiments using neuronalAPWs. We show here that similar observations can be madeusing pure populations of LVA and HVA channels, since APWstimulation of HEK cells expressing P-type channels (a1A-a

isoform; Ref. 14) induces a transient Ca21 influx overlappingthe APW duration. This Ca21 influx was ;7-fold smaller thanthe one mediated by the a1G subunit. The Ca21 influx gated bythe a1G subunit is expected to play a specific role in excitability,such as in the activation of calcium-dependent potassium chan-nels (51). In addition, it is also important to note that steadystate activation and inactivation properties strongly supportevidence for a window current that occurs in a voltage rangeclose to membrane resting potentials (265/255 mV). This sug-gests a possible involvement of a1G/T-type current in Ca21

signaling at resting membrane potential, consistent with itsrole in basal secretion of aldosterone in adrenal glomerulosacells (5). Some other cellular functions including gene activa-tion and cell proliferation, as well as critical developmentalstages and pathophysiological situations, would also depend ona background Ca21 influx through T-type channels (7, 52–54).Overall, our expression studies have clearly demonstrated thattwo specific properties of T-type channels, i.e. slow deactivationand window current at cell resting potential, can be investi-gated on human a1G-channels. Further experiments shouldtherefore explore in detail the specificities of the Ca21 signalinggenerated by the various a1G isoforms, as well as for the a1H

and a1I subunits, possibly at the molecular level in structure-function studies. A steady inward Ca21 current in the range of

cellular resting potentials is hypothesized to play an importantphysiological role, and new investigations should contribute todetermining the physiological relevance of T-type channel ac-tivity and aid in our interpretation of an increase in theirexpression during fetal life and in pathophysiology.

Acknowledgments—We gratefully acknowledge Drs. P. Berta, F. A.Rassendren, S. Richard, F. Chatail, F. Grigorescu, and C. Lautier forvaluable technical support and helpful comments. We are most gratefulto S. Spiesser for excellent technical assistance. We also thank Dr. T. P.Snutch for providing the cDNA encoding a1A subunit and Dr. M. Seagarfor critical reading of the manuscript.

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Molecular and Functional Properties of the Human alpha 1G Subunit That Forms T-type Calcium Channels

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