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J Biol Chem, Vol. 275, Issue 9, 6090-6100, March 3, 2000

Molecular and Functional Properties of the Human _1G Subunit That Forms T-type Calcium Channels^*

Arnaud Monteil^§, Jean Chemin, Emmanuel Bourinet, Gérard Mennessier^¶, Philippe Lory, and Joël Nargeot

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

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

We describe here several novel properties of the human _1G subunit that forms T-type calcium channels. The partial intron/exon structure of the corresponding gene CACNA1G was defined and several _1G isoforms were identified, especially two isoforms that exhibit a distinct III-IV loop: _1G-a and _1G-b. Northern blot and dot blot analyses indicated that _1G mRNA is predominantly expressed in the brain, especially in thalamus, cerebellum, and substantia nigra. Additional experiments have also provided evidence that _1G mRNA is expressed at a higher level during fetal life in nonneuronal tissues (i.e. kidney, heart, and lung). Functional expression in HEK 293 cells of a full-length cDNA encoding the shortest _1G isoform identified to date, _1G-b, resulted in transient, low threshold activated Ca²⁺ currents with the expected permeability ratio (I_Sr > I_Ca  I_Ba) and channel conductance (~7 pS). These properties, together with slowly deactivating tail currents, are typical of those of native T-type Ca²⁺ channels. This _1G-related current was inhibited by mibefradil (IC₅₀ = 2 µM) and weakly blocked by Ni²⁺ ions (IC₅₀ = 148 µM) and amiloride (IC₅₀ > 1 mM). We showed that steady state activation and inactivation properties of this current can generate a "window current" in the range of 65 to 55 mV. Using neuronal action potential waveforms, we show that _1G channels produce a massive and sustained Ca²⁺ influx due to their slow deactivation properties. These latter properties would account for the specificity of Ca²⁺ influx via T-type channels that occurs in the range of physiological resting membrane potentials, differing considerably from the behavior of other Ca²⁺ channels.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Transmembrane voltage-dependent calcium channels control Ca²⁺ ion entry from the extracellular space and thereby regulate various cellular processes such as muscle contraction, neuronal development and plasticity, secretion, and gene expression. Functional identification of voltage-dependent calcium channel subtypes based on their biophysical properties have distinguished low voltage-activated (LVA)¹ and high voltage-activated (HVA) Ca²⁺ currents. LVA calcium currents, mainly referred to as "T-type currents" due to their fast inactivation and small conductance, have been described in a wide variety of cell types (1). Although their physiological functions still remain obscure, they are thought to play a role in neuronal burst firing (2), pacemaker activity in heart (3, 4), aldosterone secretion (5), or fertilization (6). T-type currents are mainly detected at early stages of development as well as at a given transition (G₁/S) of the cell cycle (7-9). In addition, it has been reported that enhanced expression of T-type current is associated with a variety of diseases that include cardiac hypertrophy (10), hypertension (11), and epilepsy (12). Unfortunately, the lack of specific T-type channel blockers has considerably impaired their functional characterization. The identification of the molecular structure of T-type channels has therefore become an important challenge in order to unravel their implication in cellular functions (13).

The low level of knowledge about the molecular nature of T-type channels contrasts with the extensive molecular and functional characterization of HVA channels (L-, N-, P/Q-, and R-types) related to the discovery of specific drugs and toxins. HVA calcium channels exhibit an oligomeric structure composed of a pore-forming ₁ subunit containing voltage sensors and drug/toxin binding sites, associated with regulatory subunits (, ₂/, and ). The functional diversity of HVA calcium channels is primarily related to the existence of several ₁ subunits (_1A-F and _1S) encoded by distinct genes, many of which generate splice variants with specific properties, as described for the _1A isoforms that generate P/Q-type channels (14).

A search in the genetic data bases for sequences homologous, but not identical, to known Ca²⁺ channel ₁ subunits was an alternative to conventional molecular cloning strategies for the identification of novel ₁ subunits, and a major advance in T-type Ca²⁺ channel studies has resulted from the identification of several ESTs and genomic sequences corresponding to a subset of distantly related ₁ subunits (15). The full-length cDNAs encoding three distinct ₁ subunits (_1G, _1H, and _1I) have recently been identified, which give rise, in expression systems, to Ca²⁺ channel currents exhibiting hallmarks of native T-type currents (15-17). The genes that encode mammalian _1G, _1H, and _1I proteins are related to the Caenorhabditis elegans gene c54d2.5 described as a putative Ca²⁺ channel. These recent data (15-18) provide clear evidence that genes encoding T-type channels are members of a third subfamily in addition to the gene family encoding neuronal calcium channels (_1A, _1B, and _1E) and the gene family encoding L-type calcium channels (_1C, _1D, _1F, and _1S). An important issue now is to elucidate the molecular basis for the functional diversity of T-type Ca²⁺ channels (2).

To address this issue, we have cloned and expressed a human _1G subunit. The molecular data presented here describe several new findings concerning the _1G subunit, such as the existence of several splice variants, the partial gene structure, and the mRNA expression pattern in adult and fetal human tissues. Functional expression was achieved in order to determine the electrophysiological and pharmacological profiles of the human _1G-related currents, and we show that the _1G subunit generates a sustained Ca²⁺ current with specific features. Characterization of the human _1G subunit is an important step that significantly extends our knowledge of the molecular and functional properties of the T-type Ca²⁺ channels.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

In Silico Strategies and Probe Design-- Genetic data bases were searched for cDNA and genomic sequences homologous, but not identical, to known ₁ subunit cDNAs using (i) sequence similarity search (BLAST; Ref. 19) and (ii) a search for conserved motifs, such as a voltage sensor S4 = ((R/K)XX)₄ (BioMotif).² Among the various human sequences that were identified, we focused on sequences homologous to the C. elegans gene c54d2.5 (GenBank^TM accession no. U37548). Two EST clones, H06096 and H19230, available from the IMAGE consortium (20), were sequenced and compared with a large set of calcium channel ₁ and sodium channel  cDNAs, using the ClustalW1.7 multialignment software (21). These cDNA sequences were thus defined as candidates for putative new calcium channel ₁ subunits in humans and subsequently used as probes for both Northern blot analysis and screening of cDNA libraries. The genomic region of chromosome 17q22 that contains the CACNA1G gene was recently sequenced (AC004590). The identification of the intron/exon structure was performed using the GRAIL software (22) for the search of putative exons. Alignment of the cDNA sequences characterized in this study, together with the genomic sequence, led us to identify the exons of the CACNA1G gene that form _1G transcripts.

Isolation and Characterization of Human cDNAs-- A gt10 human cerebellum cDNA library (CLONTECH) was screened by conventional filter hybridization according to the manufacturer's protocols, using a probe generated by PCR using H06096 cDNA as template. This probe covers nucleotides 3660-4590 of the final cDNA. Hybridization was performed at 42 °C for 16-20 h in a solution containing 50% formamide using a random primed [-³²P]dCTP-labeled probe that was added at a concentration of 5 × 10⁶ cpm/ml. The membranes were then washed with a final stringency of 0.1× SSC, 0.1% SDS at 65 °C. Three gt10 clones named CC1/CC2/CC3 were identified. An additional EST (H19230) corresponding to the 3' region of the messenger was also identified. The completion of the 5' region of the sequence was achieved using long reverse transcription-polymerase chain reaction (RT-PCR) protocols (23) on mRNA from human total brain (CLONTECH) using primers defined from the rat sequence (Ref. 15; AF027984) and RACE-PCR (24). CC1 and CC3 are overlapping clones that correspond to a splice variation of the human _1G. The resulting isoforms were designated _1G-a (AF126966) and _1G-b (AF126965). The cDNA structure and sequence encoding these subunits was further identified following the characterization of two overlapping cDNA fragments of 4056 bp (nt 120 to 3936) and 3432 bp (nt 2840-6272), according to AF126965 (see Fig. 1A). Nucleotide sequences were determined using automatic sequencing (Applied Biosystems) with the dye terminator strategy. A full-length cDNA encoding the human _1G isoform designated _1G-b was constructed using the unique restriction sites mentioned in Fig. 1A and subsequently subcloned in the mammalian expression vector pBK-CMV (Stratagene).

Long Range RT-PCR for Native Isoform Identification-- A search for native isoforms of the _1G subunit was further performed using long range RT-PCR (23) and sequence examination of the cDNA fragment covering 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 _1G isoform detected. This analysis was achieved using four different sources of human mRNA: adult whole brain, adult cerebellum, adult thalamus, and fetal kidney (CLONTECH). First strand cDNA synthesis (reverse transcription) was performed using Superscript 2 (Life Technologies) with a sequence-specific primer (5'-GCTTTCTCCCAACAGCTTC-3') according to the manufacturer's protocol. The enzyme was then heat-inactivated (15 min, 70 °C). After cooling on ice, the samples were treated with ribonuclease H (Life Technologies, 4 units) for 20 min at 37 °C. PCR amplification was performed with 2 µl of the reverse transcription mixture in a final volume of 50 µl using the Expand Long Template PCR system (Roche Molecular Biochemicals) with 10× buffer 1 (10× buffer 1: 500 mM Tris-HCl, pH 9.2, 160 mM (NH₄)₂SO₄, 17.5 mM MgCl₂) supplemented with Me₂SO to a final concentration of 5%, 200 nM dNTP, 50 pmol of forward primer (5'-CTTCGGCAACTACGTGCTCTTC-3'), 50 pmol of reverse primer (5'-TCCTGAAATCCAGCTCAGCTCC-3'), and 2.6 units of DNA polymerase mix. A hot start PCR procedure was performed with the 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 cycles with denaturation at 94 °C for 10 s, annealing at 65 °C for 30 s, extension 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 pUC18 using the Sureclone ligation kit (Amersham Pharmacia Biotech). Sixty-eight positive clones were randomly chosen and fully sequenced using automatic sequencing (Applied Biosystems) with the dye terminator technology. A similar RT-PCR strategy was used for the identification of cDNA fragments covering from nt 120 to 3936 with the forward and reverse primers 5'-TAGAGCCCACCAGATGTGCC-3' and 5'-GAGATGAGCACCAACAGCCC-3', respectively. No evidence for sequence variation has been identified to date in this region.

Northern and Dot Blot Analyses-- Commercial human Northern blot membranes (CLONTECH) were hybridized using a 930-bp fragment (nt 3660-4590) generated by PCR amplification and random-primed with [-³²P]dCTP. The membranes were treated according to the manufacturer's protocol. Relative amounts of mRNA in each line were evaluated using two probes: a ubiquitin probe and a -actin probe as internal controls. Two independent dot blot membranes, normalized in their mRNA abundance by the manufacturer according to the expression profile of eight housekeeping genes (Master-blot mRNA membrane; CLONTECH) were treated in similar conditions using a probe covering the C-terminal region (nt 6440-6970). The exposure time for Northern and dot blot membranes was 6 days. Densitometric analysis of the autoradiograms was performed using an AlphaImager system (Alpha Innotech Corp.) in order to provide semiquantitation of _1G mRNA in each 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 the pBK-CMV construct that encodes for the _1G-b subunit, in a 1:10 ratio. Human embryonic kidney cells HEK-293 were grown in Dulbecco's modified Eagle's medium (Eurobio) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (v/v). For optimal transfection, cells were plated at 30-40% confluence on Petri dishes coated with poly-L-ornithine (Sigma). A standard calcium phosphate transfection procedure was performed. Positively transfected cells were identified with anti-CD8 antibody-coated beads (Dynal) and further analyzed by voltage clamp experiments. cDNAs encoding the rat brain _1A-a, ₂/_1b, and _1b subunits were inserted in the vertebrate expression vector pMT2 (14) and cotransfected in HEK 293 cells as a mix of _1A-a, ₂/_1b, _1b, and CD8 cDNAs at a molar ratio of 1:1:1:0.1.

Electrophysiology-- Macroscopic currents were recorded by the whole-cell patch clamp technique at room temperature (~21 °C) using an Axopatch 200B amplifier. Data were acquired on a PC computer using the pClAMP6 software suite (Axon Instruments). Records were filtered at 5 kHz. Leak and capacitive currents were subtracted using a P/5 procedure when needed (i.e. for tail current recordings). Extracellular solution contained 2 mM CaCl₂ (or 2 mM BaCl₂ or 2 mM SrCl₂), 160 mM TEACl, 10 mM HEPES (pH to 7.4 with tetraethylammonium hydroxide). Pipettes made of borosilicate glass with a typical resistance of 1-2 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, drugs were prepared daily in the external medium, freshly (amiloride and mibefradil) or from a stock solution (NiCl₂). The various dilutions were applied to cells by a gravity-driven homemade perfusion device, controlled by solenoid valves. Single channel recordings were done in the cell-attached mode. Sylgard coated pipettes (resistance of 7-15 mega-ohms) were filled with a solution containing 110 mM BaCl₂, 10 mM HEPES (pH to 7.2 with TEAOH). Membrane potential was reduced toward 0 mV by bathing the cells in a high potassium solution containing 140 mM potassium gluconate, 10 mM EGTA, 10 mM glucose, 1 mM MgCl₂, 10 mM HEPES (pH to 7.3 with KOH). The sampling frequency for acquisition was 10 kHz, and data were filtered at 1 kHz.

Data were analyzed using pCLAMP6 (Axon Instruments), Excel (Microsoft), and GraphPad Prism (GraphPad Inc.) software programs. Whole cell current-voltage curves were fitted using a modified Boltzmann equation as described previously (26). Activation and inactivation curves were fitted with a Boltzmann equation. Apparent dissociation constants (K_d) were obtained from the fitting of the drug dose-response curves using a sigmoidal function. Results are presented as the mean ± S.E., where n is the number of cells used.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Cloning of a Human _1G Subunit-- Several cDNA clones covering from domain II to the C-terminal region of the human _1G isoform were isolated from a cerebellum cDNA library using a probe designed from the identified EST H06096 (Fig. 1A). Additional cDNA clones that cover the entire coding region of the human _1G were identified using RT-PCR and RACE-PCR from human total brain mRNA. Finally, two overlapping cDNA fragments of 4 and 3.4 kb covering from nt 120 to 6272 were obtained by RT-PCR from human total brain mRNA to further confirm the cDNA structure of human _1G isoforms. A full-length cDNA encoding the _1G-b isoform was then constructed using the overlapping cDNAs (Fig. 1A).

View larger version (21K): [in this window] [in a new window]   Fig. 1.   Cloning and molecular properties of human 1G cDNAs. A, the cloning strategy presented under "Materials and Methods." Several partial cDNA fragments were isolated from gt10 human libraries (CC1, CC2, and CC3). Two ESTs and two PCR fragments covered the 5'- and 3'-ends. Two additional PCR fragments of 4 and 3.4 kb, according to 1G-b sequence (AF126965), were subcloned and sequenced. This latter PCR fragment was used further for 1G isoform determination as described under "Materials and Methods" and in the legend to Fig. 2. A full-length cDNA was constructed using overlapping PCR strategies as well as the unique restriction sites. An additional restriction site (BssHII) was introduced by PCR in the cDNA covering the untranslated 5' region to allow final assembly and subcloning in the pBK-CMV vector. B, the partial intron/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 1G-a and 1G-b. The ATG and TGA codons are comprised in exon 1 and exon 38, respectively. , 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. , three predicted exons that are not numbered, since they have not been detected in RT-PCR experiments.

Alignment of our cDNA sequences to the corresponding genomic region of chromosome 17q22 (AC004590), together with the use of GRAIL software (22) for the identification of putative exons in the genomic sequence, led us to identify the partial intron/exon structure of the CACNA1G gene that encodes the _1G isoforms (Fig. 1B). The CACNA1G gene covers <70 kb in the 17q22 region. A total of 41 putative exons was predicted using GRAIL software (Fig. 1B). The 5'-end of CACNA1G (~500 bp), which has been determined recently (27), is comprised within a single exon. Accordingly, this exon that also contains the first ATG codon was designated exon 1. This ATG is comprised within a consensus site for initiation of translation: AXXATGG (28). The coding region of the two human _1G subunits described later in this study is composed of 34 exons. One of these two variants which has the highest homology with the original rat sequence (15), was named _1G-a. The isoform _1G-b resulted from the use of an alternate 5'-splice donor site of exon 25 combined with the acceptor site on exon 27 (Fig. 2, A and B). Four additional exons were identified in RT-PCR experiments conducted on mRNAs from human total brain, cerebellum, thalamus, and fetal kidney. These exons encode insertions within the II-III loop (insertion e), within the III-IV loop (insertion c), and within the COOH-terminal region (insertions f and d) (Fig. 2A). In our experiments, the insertion c, which corresponded to the use of exon 26 (Fig. 2B), was found only in combination with variation b. Three other predicted exons that are included in Fig. 1B have not been retrieved experimentally to date. Sequencing of cDNA fragments obtained using long range RT-PCR experiments was performed in order to determine the potential association of the a/b, c, d, e, and f regions in _1G transcripts as well as their relative abundance in native human tissues. A total of 68 cDNA fragments obtained from human total brain, cerebellum, thalamus, and fetal kidney mRNAs was analyzed (Fig. 2C). A majority of _1G-a isoform alone, or in combination with insertion e, was found in neuronal tissues. By contrast, only the _1G-b isoform, mostly associated with insertion c, was detected in fetal kidney.

View larger version (27K): [in this window] [in a new window]   Fig. 2.   Identification of a variety of isoforms for the human 1G subunit. A, schematic structure of the 1G subunit that illustrates the localization of the a/b variation generated by alternate splicing of exon 25, as well as the positions of the insertions e, c, f, and d encoded by exons 14, 26, 34, and 35, respectively. B, description of the alternative splicing mechanisms that are expected to give rise to the 1G-a, 1G-b, and 1G-bc isoforms. Summary of the analysis of the PCR-derived cDNA fragments encoding the 3.4-3.8-kb fragment (from domain II to C terminus). A total of 68 clones deriving from total brain (22), cerebellum (22), thalamus (7), and fetal kidney (17) were analyzed by sequencing, and the occurrence of each combination is reported. Note that a truncated form of 1G (+1*) was found three times. This truncation (102 bp) removes most of the IIIS6 region of the protein.

The construct _1G-a encodes a 2250-amino acid (aa) protein with a calculated molecular mass of 249.333 Da. Primary sequences of the two variants _1G-a and _1G-b were compared with the rat and mouse _1G proteins (Fig. 3). The four transmembrane domains are highly conserved (98-100% identity). The connecting loops between domains I and II and between domains II and III, as well as the NH₂ and COOH-terminal regions are more divergent (85-95% identity). Within the II-III loop, overall identity is 90% without taking into account insertion e (23 aa), which occurs in human, rat, and mouse (Fig. 3A). The two variants _1G-a and _1G-b encode a distinct intracellular III-IV loop (Fig. 3B). By contrast with the mouse _1G sequence, insertion c (18 aa) was found associated only with the splice variant b in our experiments (Fig. 2C). Nevertheless, this intracellular loop is shorter (50-70 aa) compared with others (~300 aa). The III-IV loop in isoform _1G-a is 100% identical to the rat _1G sequence. The other isoform, _1G-b, exhibits a shorter III-IV linker resulting from a 21-bp deletion described above. Sequence comparison of the III-IV loop of several _1G, _1H, and _1I subunits, as well as the putative c54d2 gene product, revealed that sequence variability within this intracellular 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 corresponding to insertions f (48 aa) and d (45 aa) in the C-terminal region of the human _1G subunit that are presented in Fig. 3C have not been described to date. The search for putative regulatory sites, such as consensus phosphorylation sites for protein kinase A and protein kinase C, led us to identify eight protein kinase A and up to 20 protein kinase C sites contained within the intracellular regions. Most of these consensus sites are conserved among human and rat sequences. Interestingly, a protein kinase A/protein kinase C consensus site identified in loop III-IV for _1G-a is removed in the isoform _1G-b, as a consequence of splicing of exon 25 (Fig. 3B).

View larger version (50K): [in this window] [in a new window]   Fig. 3.   Multiple alignments and primary sequence analysis of the human 1G subunit. A, multiple alignments performed with ClustalW1.7 (blosum matrix) of the deduced aa sequences that encode for the intracellular loop between domain II and domain III of 1G. The sequences presented are human 1G-a (ha1Ga; AF126966); human 1G-b (ha1Gb; AF126965); human partial cDNA NBR13/1G-c (ha1G; AB012043), which carries out insertion e (+e); rat 1G isoform 1 (ra1G1 (15); AF027984); rat 1G isoform 2 (ra1G2; AF125161); mouse 1G (mA1G (30); AJ0112569). One should note that the 23-aa insertion is retrieved in human, rat, and mouse. #, insertion; *, nonconserved aa substitution; two dots:, strongly similar aa substitution; one dot, weakly similar aa substitution. B, multiple alignments performed with ClustalW1.7 of the deduced aa sequences that encode the intracellular loop between domain III and domain IV of 1G. In addition to the set of sequences defined above, the sequences encoding for the C. elegans c54d2 (U37548); human 1H isoform 1 (ha1H1 (16); AF051946); human 1H isoform 2 (ha1H2 (18); AF073931); and rat 1I (ha1I (17); AF086827) are added. Symbols are as defined for A. The symbols in the top line show the diversity of c54d2 with the mammalian sequences. The bottom line illustrates the aa diversity among the various mammalian 1 subunits encoding T-type channels. Note that a putative protein kinase A/protein kinase C phosphorylation site (#) is removed in the human isoform 1G-b (AF126965; ha1Gb). C, alignment of the C-terminal region of several human 1G isoforms that carry out no insertion (df; AF126966), insertion f (+f), or insertion d (+d), together with the mouse and rat sequences.

Distribution of _1G mRNA in Human Tissues-- Northern blot analysis of a large variety of adult human tissues showed that _1G mRNA is predominantly present in the brain (Fig. 4A). Nonneuronal tissues that were positive were ovary and placenta. A very weak band was also observed in testis, small intestine, colon, and heart. Positive tissues exhibited a major band of approximately 8.5 kb along with discrete larger bands. The reason for a smaller band in the prostate mRNA lane is currently unknown. Considering neuronal tissues, cerebellum as well as thalamus and substantia nigra displayed a strong signal (Fig. 4B). Several other tissues from the central nervous system also revealed a significant level of _1G expression, while other areas, like hippocampus, showed a weaker _1G mRNA signal. The pattern of _1G mRNA expression identified in Northern blots was mostly confirmed using dot blot analysis with densitometric quantification (Fig. 5). Two independent dot blot membranes were examined (see "Materials and Methods"). Brain tissues showed the strongest signal (thalamus > occipital lobe > cerebellum). The only difference between dot blot and Northern blot analyses concerns the spinal cord, since expression of _1G mRNA was undetectable on Northern blot, although the use of two independent internal controls, ubiquitin and -actin, suggested that the quality this mRNA sample was correct on the Northern blot membrane. Altogether, dot blot experiments revealed a level of _1G expression in good agreement with Northern blot analysis for other neuronal tissues.

View larger version (83K): [in this window] [in a new window]   Fig. 4.   Northern blot analysis of human 1G mRNAs from a variety of tissues with commercial membranes (CLONTECH) using the same probe as for the human cerebellum cDNA library screening. Internal control was performed using a ubiquitin (Ubiq.) and a -actin (not shown) probe provided by the manufacturer (CLONTECH). A, the pattern shows strong expression in brain but also in heart, placenta, and small intestine. B, the expression profile for a variety of human central nervous system regions is shown.

View larger version (27K): [in this window] [in a new window]   Fig. 5.   Dot blot analysis of adult and fetal tissues. Dot blot analysis of human 1G transcripts with commercial membranes (CLONTECH) using a probe that covers the 3'-end of the transcript (exon 38, nt 6440-6970) is shown. A, relative intensities of the spots were determined using the AlphaImager system (Alpha Innotech Corp.). B, comparison and quantitation of 1G transcripts in fetal (black bar) and adult (white bar) tissues were determined by the densitometric analysis on two independent master blot membranes (designated dot 1 and dot 2).

It is worth underlining the fact that analysis of two independent dot blot membranes revealed that the _1G mRNA expression level was significantly higher in fetal peripheral tissues, heart, kidney, lung, spleen, and thymus, compared with adult tissues (Fig. 5B). This pattern of expression was not displayed in the brain, since the amount of _1G mRNA was equivalent in adult and fetal brain. Since dot blot membranes are normalized for the amount of mRNA present in each dot (see "Materials and Methods"), we have performed a densitometric analysis that shows the relative abundance of _1G mRNA in these tissues (Fig. 5B). Altogether, these data indicate that the expression of _1G is developmentally regulated.

Electrophysiological Description of the Human _1G Currents-- Functional properties of human _1G-dependent channels were investigated using the _1G-b isoform, as expressed in HEK-293 cells. This _1G-b isoform corresponds to the minimal structure identified to date for the native human _1G subunit. Robust T-type Ca²⁺ currents were recorded in the presence of 2 mM Ca²⁺ (Fig. 6A). Activation kinetics was voltage-dependent, with time constants ranging from 10 to 1.2 ms for potentials between 50 and +30 mV. Inactivation kinetics was also strongly voltage-dependent. Current decay was best described by a single exponential function, with time constants ranging from 35 to 12 ms between 50 and +30 mV. This property implied criss-crossing of current traces as observed for native T-type currents (29).

View larger version (26K): [in this window] [in a new window]   Fig. 6.   Electrophysiological properties of the currents generated by the human 1G subunit. A, currents evoked by increasing depolarizations from 100 mV to +50 mV with 10-mV increments (from holding potential of 100 mV). The arrow indicates the peak current recorded for a test pulse to 30 mV. Note the criss-crossing pattern of current traces. B, averaged current density/voltage relationship obtained from 15 cells. C, illustration of the permeation profile of 1G channel for divalent cations. Equimolar substitution from Ca2+ to Ba2+ ions induces a slight decrease (5%) in peak current amplitude, while equimolar substitution with Sr2+ increases current amplitude (35%). D, mean Ba/Ca and Sr/Ca permeability ratios obtained from peak current amplitude values measured in 2 mM Ca2+, Ba2+, and Sr2+ (n = 6). E, single channel measurements of 1G currents recorded using a tail current protocol (see Refs. 16 and 17). The voltage protocol includes a 10-ms step to +30 mV followed by a test pulse to the indicated potentials. F, unitary current amplitudes were obtained at various potentials (120 to 0 mV) and plotted as a function of the test potential value. Linear regression revealed a slope conductance of 7.3 picosiemens (n = 3).

This current activated around 60 mV and peaked at 30 mV, while reversal potential was observed at +50 mV (Fig. 6B). When calcium (Ca²⁺) was replaced with equimolar barium (Ba²⁺) or strontium (Sr²⁺) ions, _1G currents reflected permeation properties typical of that of T-type calcium channels (Fig. 6C). The amplitude of Ba²⁺ currents was smaller (permeability ratio of Ba²⁺/Ca²⁺ ~0.96), while Sr²⁺ currents were larger (permeability ratio of Sr²⁺/Ca²⁺ ~1.35) than Ca²⁺ currents (n = 6; Fig. 6D). Interestingly, Ba²⁺ current inactivation kinetics was faster, compared with Ca²⁺ current. Single channel currents were measured in the presence of 110 mM Ba²⁺ (Fig. 6E), using tail current protocols (16). Unitary current amplitude was recorded for membrane potentials ranging from 120 to 0 mV, indicating a slope conductance of 7.3 pS (n = 3; Fig. 6F). Current amplitude at 0 mV was 0.39 ± 0.2 pA (n = 3).

Pharmacological Properties of Human _1G Subunit-- We have studied the sensitivity of human _1G channels to three molecules, nickel (Ni²⁺), amiloride, and mibefradil, that are considered to be T-type channel blockers. The divalent ion Ni²⁺ was described as a potent blocker of T-type currents; however, block of the Ca²⁺ currents generated by the human _1G subunit was modest, with an IC₅₀ of 148 ± 10 µM (n = 8; Fig. 7A). This _1G current was also poorly sensitive to amiloride, since less than 30% of inhibition in the current amplitude (n = 5) was obtained in the presence of 1 mM amiloride (not shown). Finally, mibefradil, considered as the most potent T-type channel blocker (30), affected _1G current amplitude with an IC₅₀ of 2 µM (n = 7; Fig. 7B) in good agreement with previous studies on native and recombinant T-type channels.

View larger version (15K): [in this window] [in a new window]   Fig. 7.   Pharmacological profile of 1G channels. A, mean dose-effect curve for Ni2+ (n = 8) shows an IC50 of 148 ± 10 µM. The effect of increasing Ni2+ ion concentrations on an 1G current on peak Ca2+ current amplitude is shown in the inset. The current was evoked by a 50-ms depolarization at 30 mV (stimulation rate of 0.1 Hz). Current traces from bottom to top correspond (in µM Ni2+) to control, 1, 10, 30, 100, 300, and 1000. B, the mean dose-effect curve for mibefradil indicates an IC50 of 2 ± 0.2 µM (n = 7). The inset illustrates the effect of increasing concentrations of mibefradil on 1G current evoked using the same protocol as in A. Current traces from bottom to top correspond (in µM mibefradil) to control, 0.1, 0.3, 1, 3, and 10.

Gating Properties of Human _1G Channels-- The voltage dependence of the channel activation was determined by measuring tail current amplitudes at various depolarizing pulses (Fig. 8, A and C) that fully activates _1G currents. For that purpose, duration of each pulse was adjusted to the average time-to-peak current values presented in the upper part of Fig. 8A. Normalized amplitudes of tail currents plotted as a function of the membrane potential described a biphasic smooth curve for steady state activation, which could be best fitted by the sum of two Boltzmann functions (Fig. 8C) with half activation values of V₁ = 41.8 mV and V₂ = 14.7 V (n = 5). Steady state inactivation properties were determined using a standard double pulse protocol (Fig. 8B). A 5-s prepulse (100 to 35 mV) preceded a test pulse at 30 mV. Data were fitted by a single Boltzmann function with V_0.5 = 71.6 mV (Fig. 8C). Superimposition of the steady state activation and inactivation curves revealed the existence of a window current in the range of 65 to 55 mV (Fig. 8C).

View larger version (19K): [in this window] [in a new window]   Fig. 8.   Steady state activation and inactivation of 1G calcium current. A, determination of the steady state activation was based on the measurement of tail current amplitudes according to the protocol presented in the upper part of A. For each depolarization, the tail current amplitude was measured for pulse durations adjusted from the membrane potential/time-to-peak plot shown in upper part of the figure. B, steady state inactivation was estimated from the measurement of the current amplitude at 30 mV after a 5-s predepolarization pulse of increasing amplitude (100 to 35 mV, 5-mV increment). C, normalized steady state activation obtained from the protocol described in A (open symbols, n = 5). The smooth curve corresponds to the best fit obtained using two Boltzmann functions with normalized activation (m) being a function of the test potential (V) according to the equation m = A1/1 + exp((V1  V)/K1) + A2/1 + exp((V2  V)/K2), where A1 and A2 are the respective contributions of each Boltzmann distribution; V1 and V2 are the two half-activation values; and K1 and K2 are the slopes. Values from the fit are A1 = 57%, A2 = 43%, V1 = 41.8 mV, V2 = 14.7 mV, K1 = 5.1 mV, and K2 = 11.9 mV. Mean steady state inactivation curve (filled symbols, n = 10) was best fitted with a single Boltzmann distribution with a half-inactivation potential (V0.5) of 71.6 mV and a slope of 4.33 mV. Superimposition of steady state activation (m) and inactivation (h) curves revealed a window current near 65 to 55 mV.

Deactivation Properties of _1G-related Channels-- The deactivation properties of human _1G channels were deduced from tail current analysis (Fig. 9). Fig. 9A shows that kinetics of tail currents was slower when repolarizing membrane potentials were more positive, reflecting a strong voltage dependence of the deactivation process for _1G-related currents (Fig. 9B). Additional experiments using action potential waveforms (APWs) as a voltage clamp command were designed to evaluate whether _1G-related channels allow brief or sustained entry of Ca²⁺ in the range of physiological membrane resting potentials (about 75 mV). When HEK cells overexpressing the human _1G subunit were stimulated with neuronal APWs (2-ms duration) applied from a holding potential of 75 mV, a slow inward Ca²⁺ current following the APW was recorded (Fig. 9C). This current was totally suppressed by the application of 1 µM mibefradil (not shown). By contrast, application of this APW protocol to cells that expressed HVA current generated by _1A subunit resulted in a transient inward calcium current that correlates with the duration of the APW (Fig. 9C). A similar result was obtained using native neurons expressing only HVA currents (not shown).

View larger version (16K): [in this window] [in a new window]   Fig. 9.   Deactivation properties of 1G currents and APW voltage clamp experiments. A, 1G currents were activated by a 7-ms test pulse at 30 mV from a holding potential of 100, and a family of tail currents was recorded for subsequent repolarizations from 120 to 40 mV. B, plot of mean deactivation time constants as a function of membrane repolarization potential (n = 5). C, Ca2+ currents generated by the 1G and 1A subunits (see labels) in response to a neuronal APW (2-ms duration; holding potential of 75 mV). Note that, according to the slow deactivation kinetics of the currents generated by the 1G subunit, activation and peak of the Ca2+ current occurred during repolarization. This inward current persisted for more than 10 ms after repolarization. In contrast, HVA current generated by the 1A-a subunit using the same APW protocol evoked a transient Ca2+ current that matches the APW duration.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

This study is the first description of the molecular and the functional properties of a cloned _1G T-type Ca²⁺ channel subunit from humans. Specific findings include (i) the description of several _1G isoforms generated by alternative splicing, (ii) evidence for a developmental regulation of _1G transcripts, and (iii) the description of specific functional properties related to slow deactivation and the existence of a window current in the range of the resting membrane potential.

Our study provides the first evidence for alternatively spliced isoforms of the _1G subunit, revealing therefore that the molecular diversity of T-type channels not only relies on the expression of three subunits encoded by distinct genes, _1G, _1H, and _1I (17). In humans, the gene CACNA1G encoding _1G is localized on chromosome 17q22 (15). Exon prediction using GRAIL (22) led us to identify 41 putative exons within the genomic sequence that covers the coding region of that gene (AC004590). We have numbered 38 of them that were unambiguously identified in cDNA cloning and RT-PCR experiments. Whether additional exons exist cannot be excluded. Recently, Toyota et al. (27) have performed 5'-RACE analysis of CACNA1G in order to identify the transcription start site of this gene in humans (GenBank^TM accession no. AF124351). In good agreement with our results, no additional exon was found upstream to the one identified here as exon 1. More importantly, these authors have demonstrated that the CACNA1G gene is found inactivated by aberrant methylation in several human tumors.

We have identified here that the _1G-b isoform corresponds to the minimal structure of the native _1G subunit in humans, and therefore it is important to consider that analysis of this isoform provides a basis for the comparison of the deduced aa sequences of _1G subunits among species, as well as the identification of regions that are critical to explore for further structure-function studies. Indeed, a mouse _1G subunit recently described (31) exhibits two insertions within loops II-III and III-IV, compared with rat _1G (15). We demonstrate here that the insertion in loop II-III corresponds to the use of an additional 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. In addition, we have identified two novel insertions d and f in the C-terminal region of the human _1G subunit. The occurrence of these latter insertions is low: 1 and 7 out of 68 for d and f, respectively. The functional role of these insertions in II-III loop and the C terminus remain to be determined. Unexpectedly, while sequencing our batch of 3.4/3.8-kb fragments that covered from domain II to the C-terminal region, we have found a truncated form of _1G cDNA (3 out of 68) that corresponds to a 102-bp deletion (nt 4498-4601; AF126965) of a region that encodes the IIIS6 segment. The reason for the existence of such a truncated form of _1G, which corresponds to an aberrant alternative splicing of exon 25, is unclear at the present time.

The differences among human, mouse, and rat _1G subunits within the III-IV loop are likely to correspond to the combination of two mechanisms. First, we have demonstrated here that the use of an alternate splice site within exon 25 can generate the two human _1G isoforms, _1G-a and _1G-b. We referred here to these isoforms as _1G-a and _1G-b, by comparison to the original rat _1G subunit (AF027984), which is identical to the human _1G-a. A similar mechanism is likely to occur in rodents, since a sequence similar to the III-IV loop of _1G-b can be retrieved in rat (32). Second, human, mouse, and rat exhibit an additional insertion, designated c, in this region (31, 32). In our study, insertion c was found only in combination with b, resulting in _1G-bc isoforms that were mostly expressed in fetal kidney. Again, the role of insertion c in the human _1G subunit has yet to be determined. Altogether, we describe here two mechanisms that are able to generate _1G isoforms with a distinct III-IV loop. The isolation and tissue distribution of cDNAs encoding the various human _1G isoforms as well the investigation of their functional properties is now an important issue, as demonstrated for the _1A isoforms that give rise to P/Q Ca²⁺ channel subtypes (14).

A common feature of the human and rodent _1G subunits, as well as for the _1H and _1I subunits, is the absence of the -subunit binding site, called AID (33), found in every HVA ₁ subtype. Such data are consistent with previous experiments showing that -subunit knock-down using antisense strategies does not affect T-type currents (34, 35). Similarly, the G protein binding sites described in the non-L ₁ subunits (36) are not retrieved. This is also the case for the Ca²⁺ binding and calmodulin binding domains involved in calcium dependent inactivation (37, 38). Several putative phosphorylation sites for protein kinase A, protein kinase C, and CaM-kinase II occur in the sequence, but their functional relevance needs to be investigated. Interestingly, the shortest isoform, _1G-b, lacks a putative protein kinase A/protein kinase C phosphorylation site in the III-IV loop.

Based on the _1G-a isoform, the aa sequence of the III-IV loop is highly conserved among the three subunits _1G, _1H, and _1I, which would suggest a conservation in function (75%; Ref. 17). It was proposed that this connecting loop might be important for inactivation of T-type channels (17, 39) on the basis of that described for Na⁺ channels (40). However, although the various _1G isoforms exhibit important sequence variations in the III-IV loop, inactivation properties of currents related to the human _1G-b subunit did not show significant differences from those of currents related to rodents _1G isoforms (15, 31). Further work is needed to clearly examine whether this region of _1G influences the voltage-dependent inactivation process and, subsequently, if sequence variations within the III-IV loop have functional consequences on T-type Ca²⁺ channel activity.

Northern blot and dot blot analyses have clearly indicated that the highest level of _1G expression occurs in human brain tissues, particularly in thalamus, cerebellum, substantia nigra, and frontal and occipital lobes. Overall, these data correlate well with those of the recent study by Talley et al. (41). Expression of _1G in thalamus is expected to underlie the low threshold spike activity described in thalamic neurons (42). Furthermore, overexpression of T-channels in this structure has been observed in GAERS rats, an animal model of absence epilepsy (12). More importantly, our data have revealed that the _1G subunit expression is developmentally regulated. While _1G is rather similarly expressed in adult and fetal brain, it is much more predominant in fetal peripheral tissues such as heart, kidney, or lung. Enhanced expression of an ₁ subunit encoding T-type channels in fetal heart correlates well with electrophysiological data, since it is generally accepted that T-type currents are preferentially recorded in embryonic or newborn myocytes (43). It complicates, however, the identification of the so-called cardiac T-type channel, since another member of the T-type channel family, _1H, has been cloned from heart tissue (16). The relative expression of both _1H- and _1G-related channels in cardiovascular tissues is therefore an important issue that needs to be further investigated.

The electrophysiological and pharmacological properties of human _1G currents were examined in a physiological external calcium concentration (2 mM), except for the determination of the single channel conductance (~7 picosiemens). Overall, biophysical properties such as low activation threshold (<60 mV), potential for peak current (30 mV), activation and inactivation kinetics, cross-over of current traces, and permeation properties (I_Sr > I_Ca  I_Ba) are typical of that of T-type currents recorded in native cells. Our data are also in good agreement with those of other cloned _1G channels, although comparison is impaired by the differences in experimental conditions. For instance, threshold of activation at more positive potentials (50 mV) and a shift of the I/V curve (peak at 10 mV) reported for the mouse isoform (30) is likely to be due to the use of high concentrations (20 mM) of divalent cations. It is worth noting that steady state activation is best described by the sum of two Boltzmann distributions. Similar results were obtained for human _1H channels (18) and rat _1G channels (44). A kinetic model that includes several closed state transitions prior to opening was proposed in Ref. 44, and this scheme can accurately account for most of the qualitative and quantitative features of human _1G channels described here. By contrast, a single Boltzmann function fits the steady state inactivation. This dual behavior for activation and inactivation is reminiscent of that reported for several other channel types, including N-type in dorsal root ganglion neurons (45), for which a bimodal activation process (i.e. "willing"/"reluctant" modes) was coupled to only one inactivation process.

The pharmacological profile of human _1G-related channels was determined using the few molecules, nickel (Ni²⁺), mibefradil, and amiloride, which have been used to discriminate LVA T-type calcium channels from HVA calcium channels. The human _1G channel has a relatively high sensitivity to mibefradil with an IC₅₀ in the micromolar range. This value is quite comparable with the mibefradil inhibition of human _1H currents (1 µM) recorded in the presence of 15 mM Ba²⁺ (18). Amiloride seems to affect differentially _1G- (IC₅₀ > 1 mM) and _1H-related currents (IC₅₀ = 167 µM; Ref. 18), although the differences in the recording conditions should again be considered. Similarly, we found a rather low sensitivity to Ni²⁺ ions of human _1G currents (IC₅₀ = 150 µM), as compared with _1H calcium channels for which an IC₅₀ of 6 µM was reported (18). One should note that the mouse _1G channel is blocked by an even higher Ni²⁺ concentration (IC₅₀ > 1.1 mM in 20 mM Ca²⁺; Ref. 31). Nevertheless, discrepancies in the sensitivity to Ni²⁺ ions are consistent with the data reported on native cells (2). It is important to note that native T-type channels from brain regions expressing a high level of _1G subunit exhibit low sensitivity for Ni²⁺ ions in good agreement with that described for expressed _1G channels (2, 41, 46). A consequence of these data is that the block by micromolar concentrations of Ni²⁺ should not longer be considered as distinguishing LVA from HVA channels (47).

A hallmark of T-type channels is the slow kinetics of their current deactivation, and our data clearly show that the deactivation kinetics of the Ca²⁺ current related to human _1G channels is voltage-dependent. We further demonstrate that the consequence of a slow deactivation process can be visualized using neuronal APWs as a voltage clamp command. Overexpression of T-type calcium channel in HEK cells greatly improves such investigations, and we show that APW stimulation (<2 ms) from cell's resting potential values (75 mV) induces a sustained Ca²⁺ current that occurs during repolarization and persists for more than 15 ms. The kinetics of this Ca²⁺ current is consistent with the kinetics of tail currents evoked by a square-pulse with membrane repolarization at this potential. As expected, this Ca²⁺ influx is likely to be shaped by the pattern of APWs, such as duration, resting membrane potential, and firing rate (48). Deactivation is a feature that distinguishes LVA/T-type currents ( ~ 5 ms; Ref. 2) from HVA currents ( ~ 0.3 ms; Ref. 49). McCobb and Beam (50) have reported that distinct Ca²⁺ signals are generated by LVA and HVA channels in voltage clamp experiments using neuronal APWs. We show here that similar observations can be made using pure populations of LVA and HVA channels, since APW stimulation of HEK cells expressing P-type channels (_1A-a isoform; Ref. 14) induces a transient Ca²⁺ influx overlapping the APW duration. This Ca²⁺ influx was ~7-fold smaller than the one mediated by the _1G subunit. The Ca²⁺ influx gated by the _1G subunit is expected to play a specific role in excitability, such as in the activation of calcium-dependent potassium channels (51). In addition, it is also important to note that steady state activation and inactivation properties strongly support evidence for a window current that occurs in a voltage range close to membrane resting potentials (65/55 mV). This suggests a possible involvement of _1G/T-type current in Ca²⁺ signaling at resting membrane potential, consistent with its role in basal secretion of aldosterone in adrenal glomerulosa cells (5). Some other cellular functions including gene activation and cell proliferation, as well as critical developmental stages and pathophysiological situations, would also depend on a background Ca²⁺ influx through T-type channels (7, 52-54). Overall, our expression studies have clearly demonstrated that two specific properties of T-type channels, i.e. slow deactivation and window current at cell resting potential, can be investigated on human _1G-channels. Further experiments should therefore explore in detail the specificities of the Ca²⁺ signaling generated by the various _1G isoforms, as well as for the _1H and _1I subunits, possibly at the molecular level in structure-function studies. A steady inward Ca²⁺ current in the range of cellular resting potentials is hypothesized to play an important physiological role, and new investigations should contribute to determining the physiological relevance of T-type channel activity and aid in our interpretation of an increase in their expression during fetal life and in pathophysiology.

	ACKNOWLEDGEMENTS

We gratefully acknowledge Drs. P. Berta, F. A. Rassendren, S. Richard, F. Chatail, F. Grigorescu, and C. Lautier for valuable technical support and helpful comments. We are most grateful to S. Spiesser for excellent technical assistance. We also thank Dr. T. P. Snutch for providing the cDNA encoding _1A subunit and Dr. M. Seagar for critical reading of the manuscript.

	FOOTNOTES

^* This work was supported in part by the Program Génome du CNRS, Association pour la Recherche contre le Cancer Grant ARC9011, and Association Française contre les myopathies.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EMBL Data Bank with accession number(s) AF126966 (_1G-a) and AF126965 (_1G-b).

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

To whom correspondence should be addressed. Tel.: 33 499 61 99 36; Fax: 33 499 61 99 01; E-mail: philippe.lory@igh.cnrs.fr.

² G. Mennessier, submitted for publication.

	ABBREVIATIONS

The abbreviations used are: LVA, low voltage activated; HVA, high voltage activated; EST, expressed sequence tag; RACE, rapid amplification 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).

	REFERENCES

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				P. Mariot, K. Vanoverberghe, N. Lalevee, M. F. Rossier, and N. Prevarskaya Overexpression of an alpha 1H (Cav3.2) T-type Calcium Channel during Neuroendocrine Differentiation of Human Prostate Cancer Cells J. Biol. Chem., March 22, 2002; 277(13): 10824 - 10833. [Abstract] [Full Text] [PDF]

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				O. Lesouhaitier, A. Chiappe, and M. F. Rossier Aldosterone Increases T-Type Calcium Currents in Human Adrenocarcinoma (H295R) Cells by Inducing Channel Expression Endocrinology, October 1, 2001; 142(10): 4320 - 4330. [Abstract] [Full Text] [PDF]

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				G. Wennemuth, R. E. Westenbroek, T. Xu, B. Hille, and D. F. Babcock CaV2.2 and CaV2.3 (N- and R-type) Ca2+ Channels in Depolarization-evoked Entry of Ca2+ into Mouse Sperm J. Biol. Chem., July 7, 2000; 275(28): 21210 - 21217. [Abstract] [Full Text] [PDF]

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				C. Berthier, A. Monteil, P. Lory, and C. Strube {alpha}1H mRNA in single skeletal muscle fibres accounts for T-type calcium current transient expression during fetal development in mice J. Physiol., February 1, 2002; (2002) 200101324. [Abstract] [PDF]

				J. Chemin, A. Monteil, E. Perez-Reyes, E. Bourinet, J. Nargeot, and P. Lory Specific contribution of human T-type calcium channel isotypes (1G, 1H and 1I) to neuronal excitability J. Physiol., February 15, 2002; (2002) 200101326. [Abstract] [PDF]

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