Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

Voltage-gated calcium channels mediate Ca²⁺ influx in excitable cells. Upon depolarization of the plasma membrane, a calcium channel undergoes a series of conformational changes initiated by the charge movement, which results in the opening of a pore or conductance pathway selective for the influx of calcium ions. Calcium channels are a diverse class of proteins that, based on their electrophysiological and pharmacological properties, have been traditionally classified into types L, T, N, P, Q, and R (for review, see Catterall, 2000). Except for the T-type calcium channel, which is a low-voltage-activated channel, the L-, N-, P-, Q-, and R-type are all high-voltage-activated channels, which are normally activated above 40 mV. A calcium channel is a multisubunit protein complex (Catterall, 2000) that is composed of a pore-forming ₁ subunit (Perez-Reyes et al., 1989) and three regulatory subunits: ₂, , and . All of these subunits from rabbit have been characterized by molecular cloning (Tanabe et al., 1987; Ellis et al., 1988; Ruth et al., 1989; Jay et al., 1990). The ₂ and  subunits regulate almost all aspects of channel properties and increase the functional channel density on the cell surface (Lacerda et al., 1991).

To date, at least 10 different types of calcium channel 1 subunits (Catterall 2000), four types of  subunits (for review, see Birnbaumer et al., 1998), three types of ₂ subunits (Ellis et al., 1988; Klugbauer et al., 1999; Gao et al., 2000), and five types of  subunits (Jay et al., 1990, Letts et al., 1998, Klugbauer et al., 2000) have been cloned and characterized. However, pharmacological and electrophysiological studies have identified more subtypes of voltage-gated calcium channels in excitable cells than the type of ₁ subunits currently known. Therefore, either more 1 subunits and/or their splicing variants are present or the ₂, , and  subunits also contribute to the pharmacological and electrophysiological diversity of calcium channels. In the later case, the diverse calcium channels are formed from different combinations of ₁, ₂, , and  subunits.

The calcium channel ₂ subunit is a heavily glycosylated protein that is encoded by a single gene and post-translationally cleaved to yield 2 and  subunits linked by a disulfide bond (De Jongh et al., 1990) with a single transmembrane segment (Gurnett et al., 1996; Wiser et al. 1996; Felix et al., 1997). The ₂ subunit regulates many functional aspects of calcium channels, such as gating, regulating voltage dependent kinetics, and increasing functional channel density on the plasma membrane (Shistik et al., 1995; Bangalore et al., 1996; Qin et al., 1998; Shirokov et al., 1998; Sipos et al., 2000). In addition to its regulatory functions, the ₂ subunit is also a site for ligand binding. Recently, the novel anticonvulsant drug, gabapentin (1-aminomethyl cyclohexane acetic acid), was shown to bind with high affinity directly to the calcium channel ₂-1 subunit (Gee et al., 1996; Brown and Gee, 1998). This binding affects neuronal excitability by modifying calcium channel activity (Rock et al., 1993), which may be the drugs' underlying mechanism in controlling neuropathic pain (Field et al., 2000).

Analyses of the Drosophila melanogaster genome have revealed four ₁, three ₂, one , and  subunit for calcium channels (Littleton and Ganetzky, 2000). Based on the ratio of ₁ to ₂ (4 to 3) in D melanogaster, we proposed that more than three types of ₂ subunits must exist in mammals, although several alternative splicing variants of ₂-1 and ₂-2 subunits have been identified (Kim et al., 1992; Gilad et al., 1995; Angelotti and Hofmann 1996; Klugbauer et al. 1999; Hobom et al., 2000). In addition, no regulatory subunits of T-type channels (_1G to _1H) have yet been identified, further suggesting that additional calcium channel regulatory subunits may exist (Lacinova et al., 1999). Here, we report the molecular cloning of a putative novel ₂ subunit, ₂-4. The ₂-4 gene contains 39 exons spanning about 130 kb and is colocalized with a calcium channel ₁ Ca_V1.2 gene on human chromosome 12. Alternatively spliced products were identified by molecular cloning and confirmed by genomic sequence analysis. Studies of tissue distribution by Northern analysis and immunohistochemistry suggest that the ₂-4 subunit is highly expressed in some endocrine cells.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Rapid Amplification of cDNA Ends Coupled to Polymerase Chain Reaction. To clone the full-length human calcium channel ₂-4 subunit, RACE-PCR followed by Nest-PCR was used. For both 5' and 3' RACE-PCR, several primers were synthesized based on the sequence of expressed sequence tag clone AA001473. Reverse primers, A2-4-9 (5'-CAG GGG CTG GGC TGC ACT GTG GTG GTG-3') and A2-4-10 (5'-CTC TCG GGA CCT CTT GGA GAT CAG AAT-3') were used for RACE-PCR of the 5' end, and forward primer, A2-4-42 (5'-AGC ATG GGG GTG TTC AGC CAA GTG ACT-3') was used for RACE-PCR of the 3'-end. Primary RACE-PCR was performed in a 50-µl final volume. The reaction mixture contained 5 µl of Marathon-Ready human brain cDNA purchased from BD Clontech (Palo Alto, CA), 5 µl of 10 × reaction buffer, 200 µM dNTP, 200 nM AP1 primer (BD Clontech), 200 nM specific primer A2-4-9, and 1 µl of 50 × Advatage2 DNA polymerase mixture (BD Clontech). The thermal cycler parameter for RACE-PCR was: initial denaturing at 94°C for 30 s, five cycles of 94°C/5 s and 72°C/4 min, five cycles of 94°C/5 s and 70°C/4 min, and 20 cycles of 94°C/5 s and 68°C/4 min. After RACE-PCR reaction, the nest PCR was performed to further enhance the amplification. The reaction mixture (in 50-µl final volume) contained: 5 µl of the RACE PCR product, 5 µl of 10× reaction buffer, 200 µM dNTP, 200 nM AP2 primer (BD Clontech), 200 nM specific primer A2-4-10, and 1 µl of 50× Advantage2 DNA polymerase mixture (BD Clontech). The thermal cycler parameters for the nest PCR reaction were identical to that of RACE-PCR. The nested PCR product was then subcloned with a TA Cloning kit (Invitrogen, Carlsbad, CA) and sequenced.

Assembly of Full-Length Human Calcium Channel ₂-4 Subunit. The full-length human calcium channel ₂-4 subunit was assembled and subcloned into pAGA3 vectors (Qin et al., 1997) according to standard molecular biology methods. Briefly, a 1.26-kb N-terminal fragment was subcloned into the pAGA3 vector. Two C-terminal fragments were first assembled in pBlueScript KS⁺ (Stratagene, La Jolla, CA), and the resulting 2.1-kb DNA fragment excised was then subcloned into pAGA3 containing the N terminus of h₂4 to generate the full-length human calcium channel ₂-4 subunit, pAGA3/h₂-4. The final construct was confirmed by DNA sequencing. For expression in mammalian cells, the full-length cDNA was also subcloned into pcDNA3.1 expression vector (Invitrogen).

Generation of Polyclonal Antibodies. Two peptide sequences derived from the human calcium channel ₂-3 and ₂-4 subunits were selected to raise polyclonal antibodies in rabbits. The peptides were: Ac-KVSDRKFLTPEDEASVC-amide for ₂-4 (737-752 amino acids) and Ac-QLTNQDFLKAGDKENI-amide for ₂-3 subunit (745-760 amino acids). The peptides were synthesized, and antibodies were raised and purified by BioSource International, Inc. (Camarillo, CA). The antibodies were tested by ELISA against the antigen peptides and affinity purified with the same peptides. The affinity-purified antibodies were used for immunoanalysis.

Northern Blot Analysis of the Human Calcium Channel ₂-4 Subunit Expression. The cDNA fragment encoding residues 1-270 of the human ₂-4 subunit was used as a probe. To label the probe, 25 ng of the DNA fragment was denatured in a final volume of 45 µl at 99°C for 4 min. The denatured DNA probe was incubated with 5 µl of [-³²P]dCTP at 6000 Ci/mmol (Amersham Biosciences, Piscataway, NJ) and then transferred to the tube containing a Ready-To-Go DNA Labeling Bead (-dCTP) (Amersham Biosciences) and incubated at 37°C for 30 min. The labeled probe was then separated from free [-³²P]dCTP using a MicroSpin G-50 column (Amersham Biosciences). The labeled probe was denatured at 99°C for 4 min and immediately placed on ice before being added to the hybridization solution.

In Vitro Translation. The full-length cDNA of human calcium channel ₂ subunits were first subcloned into a pAGA3 vector, which were engineered for high efficiency of in vitro transcription and translation as described in Qin et al. (1997). In vitro translation of the human calcium channel ₂ subunits were performed with TnT T7 Quick Coupled Transcription/Translation System (Promega, Madison, WI) following the vendor-recommended protocol. Briefly, 0.5 µg of ₂ constructs were added to 40 µl of TNT Quick Master Mix with 1 µl of [³⁵S]methionine (1000 Ci/mmol at 10 mCi/ml) in a final volume of 50 µl. The reaction mixtures were incubated at 30°C for 90 min. Reaction mixture (5 µl) was mixed with an equal volume of SDS/PAGE loading buffer and subjected to 4 to 12% SDS/PAGE analysis. After electrophoresis, the gels were dried for radioautography or transferred to nitrocellulose for further Western blot analysis.

Immunohistochemistry. Commercial human checkerboard tissue slides (DAKO, Carpinteria, CA; Biomeda, Foster City, CA; Novagen, Milwaukee, WI) were deparaffinized, hydrated, and processed for routine immunohistochemistry as described previously (D'Andrea et al., 1998). Briefly, slides were microwaved in Target buffer (DAKO), cooled, placed in distilled water and then treated with 3.0% H₂O₂ for 10 min. Afterward, the slides were rinsed in phosphate-buffered saline (PBS), pH 7.4, processed through an avidin-biotin blocking system according to the manufacturer's instructions (Vector Labs, Burlingame, CA), and then placed in PBS. All subsequent reagent incubations and washes were performed at room temperature. Normal blocking serum (Vector Labs) was placed on all slides for 10 min. After briefly rinsing in PBS, primary antibody (affinity-purified anti-human ₂-4 polyclonal antibodies, 1:1000 dilution) was placed on slides for 30 min. The slides were washed and a biotinylated secondary antibody, goat anti-rabbit was placed on the tissue sections for 30 min (Vector Labs). After rinsing in PBS, the horseradish peroxidase-avidin-biotin complex reagent (Vector Labs) was added for 30 min. Slides were washed and treated twice with the chromogen 3,3'-diaminobenzidine (Biomeda) for 5 min each, then rinsed in dH₂O and counterstained with hematoxylin. A monoclonal antibody to vimentin, the widely conserved, ubiquitous intracellular filament protein, was used as a positive control to demonstrate tissue antigenicity and control reagent quality. The negative controls included replacement of the primary antibody with preimmune serum or with the same species IgG isotype nonimmune serum (Vector Labs).

Transient Transfection and Cell Membrane Preparation. COS-7 cells were cultured in DMEM with 10%FBS at 37°C in 5% CO₂ incubator. One day before transfection, about 5 × 10⁶ COS-7 cells were seeded on 150-mm culture dishes. The cells were transfected with 20-µg DNA constructs using SuperFect transfection reagent (QIAGEN, Valencia, CA) following the protocol provided by the company. Membrane preparation was carried out at 4°C. Forty-eight hours after transfection, cells were washed with PBS and suspended in lysis buffer (10 mM HEPES, pH 7.4, and proteinase inhibitor cocktail). The cells were incubated on ice for 30 min followed by brief sonication. The cell debris was removed by centrifugation at 1,000g for 10 min, and the resulting supernatant was centrifuged for 30 min at 50,000g. The pellet was resuspended in the lysis buffer and kept at 80°C for Western blot analysis and gabapentin binding assay.

Tissue Preparation and Protein Extraction. The tissue preparation and protein extraction were carried out by ResGen. Approximately 220 mg of each sample was finely chopped and placed in a microcentrifuge tube. Radioimmunoprecipitation assay buffer (150 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholate, 0.5% SDS, and 50 mM Tris, pH 8.0) with protease inhibitors was added, and each sample was homogenized until the mixture was of a uniform consistency. Each sample was heated at 100°C for 10 min. Each sample was then centrifuged at 10,000 rpm for 10 min. The supernatants were removed and placed in clean tubes. The supernatants were centrifuged at 13,200 rpm for 30 min. The total protein concentration was determined by absorption at 280 nm.

Western Blot. Proteins in the gel were transferred to nitrocellulose membrane at 25 V for 60 min. After being blocked with 2% fat dry milk in 0.5% Tween 20, 100 mM NaCl, and 10 mM Tris-HCl, pH 7.4, for 1 h at room temperature, the blot was then incubated with primary antibodies in 2% fat dry milk/0.5% Tween 20, 100 mM NaCl, and 10 mM Tris-HCl, pH 7.4, at 4°C overnight. The following primary rabbit polyclonal antibodies were used: anti-₂-1 (1:200 dilution) and anti-_1C (1:200) purchased from Alomone Labs, anti-₃ (1:200) purchased from Sigma, anti-₂-3 (1:200 dilution), and anti-₂-4 (1:500 dilution; see Generation of Polyclonal Antibodies). Secondary goat anti-rabbit IgG conjugated with horseradish peroxidase 1:10,000 (from Pierce) was incubated for 1 h at room temperature. Finally, the signals were visualized on X-ray film using the ECL Plus kit (Amersham). Because Anti-HA monoclonal antibody (Roche Diagnostics) was conjugated with peroxidase, the blot was directly treated with ECL-plus kit without using secondary antibody.

Coimmunoprecipitation. Coimmunoprecipitation was carried out with cell lysates from transiently transfected HEK 293 cells by Ca_V1.2 (_1C), ₃, and ₂-4 tagged with HA epitope constructs. Two days after the transfection, the cells (from two 150-mm plates) were harvested and washed with PBS. The cells were then resuspended in 2 ml of detergent extraction buffer: 1% (v/v) Nonidet P-40, 0.5% deoxycholate, 150 mM NaCl, 5 mM EDTA, 50 mM Tris-HCl, pH 8.0, and 20 µl of protease inhibitor cocktail (Sigma), followed 10 passages each through 20- and 26-gauge needles. Cell extracts were cleared by centrifugation. Coimmunoprecipitation was carried out with 500 µl of the cell lysates in the presence of either 50 µl of anti-HA (rat monoclonal antibody) Affinity Matrix (Roche Diagnostics, Indianapolis, IN) or 50 µl of anti- Ca_V1.2 polyclonal antibody (Alomone Labs, Jerusalem, Israel)/50 µl of protein A Sepharose CL-4B (Pharmacia, Peapack, NJ). After overnight incubation at 4°C, the beads were precipitated by a brief centrifugation, and washed three times with 1 ml of lysis buffer. Finally, an equal volume of 2× SDS loading buffer was added, and 20 µl was subjected to 4 to 20% SDS. The coprecipitated subunits were then analyzed by Western blot with the indicated antibodies.

Calcium Influx Assay. HEK 293 cells were seeded in six-well plates (1.5 × 10⁵/well) the day before the transfection. The cells were transfected with 1 µg of DNA constructs as indicated by using Genejammer transfection reagent (Stratagene) following the protocol provided by the company. After 24 h, the transfected cells were replated into a 96-well plate (2 × 10³/100 µl/well) and incubated at 37°C/5% CO₂ for another 24 h.

Binding Assay. The gabapentin binding assay was based on the protocol developed by Gee et al. (1996). The binding assay was carried out in a final volume of 200 µl containing 40 µg of cell membrane, 20 nM [³H]gabapentin (127 Ci/mmol), and 10 mM HEPES buffer, pH 7.4. After incubation at room temperature for 1 h, the reaction mixture was filtered onto prewetted GF/C membranes and washed four times with ice-cold saline. The filters were then dried and counted in a liquid scintillation counter.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

Identifying and Cloning A Novel Human ₂ Subunit. The key words "Calcium Channel" were used to search the GenBank nonredundant DNA database. Twenty-nine hits were identified as related to ₂ subunits. Further sequence analysis led to the identification of two overlapping expressed sequence tag clones that might encode a novel human calcium channel ₂ subunit, with GenBank accession numbers AA001473 (572 bp in length) and H86016 (306 bp in length). The two clones were almost 100% identical in the 292-bp overlapping region, suggesting that they might arise from the same gene. The amino acid sequence derived from the longer clone, AA001473, was 40% identical to the mouse calcium channel ₂-3 subunit over residues 839 to 977 (GenBank accession number AJ010949), 36% identical to the human calcium channel ₂-2a subunit over residues 870 to 949 (GenBank accession number AF042793), and 34% identical to the human calcium channel ₂-1 subunit from residues 836 to 927 (GenBank accession number NM_000722), suggesting that it may encode a novel ₂. We used the RACE-PCR to clone its full-length cDNA and confirmed by reverse transcription-PCR.

View larger version (61K): [in this window] [in a new window]   Fig. 1.   Alternative splicing of amino and carboxyl termini of human 2-4 subunit. A and B, nucleotide and deduced amino acid sequences of two different amino termini. The amino terminus encoded by exon 1 (A) consists of a putative signal peptide, whereas the one encoded by exon 1B (B) does not. C and D, nucleotide and deduced amino acid sequences of two different carboxyl termini encoded by exons 38 (C) and 37L (D). The putative hydrophobic membrane-spanning domains are highlighted.

View larger version (28K): [in this window] [in a new window]   Fig. 2.   Human 2-4 subunit gene (CACNA2D4) structure. Human CACNA2D4 gene is composed of 36 invariant exons (exons 2-37) and four alternative exons (exons 1, 1B, 37L, and 38) that span about 130 kb of human chromosome 12p13.3. Exon 37L is continuously extended from exon 37 with an in-frame stop codon. Exons 1 and 1B both have an in-frame start codon, and exon 38 has an in-frame stop codon, indicating that there are four possible alternative splicing variants. The human calcium channel 2-4a subunit is encoded by exons 1 and 38 with 37 invariant exons (2-37), whereas human 2-4b is encoded with alternative exons 1B and 2 to 38. Another two putative splicing variants are 2-4c (exons 1-37L) and 2-4d (exons 1B and 2-37L).

View larger version (64K): [in this window] [in a new window]   Fig. 3.   Comparison of four types of human 2 subunits. A, amino acid sequences alignment of human 2-1 (GenBank accession number NM_000722), human 2-2 (GenBank accession number AF042793), human 2-3 (GenBank accession number AF516696) and human 2-4 (GenBank accession number AF516695). Regions that are identical and highly homologous in all four subunits are shaded. All 15 conserved cysteine residues in four subunits and breaking point between 2 and  subunits are indicated by asterisks or an arrow, respectively. Four regions of 2-1 subunit required for gabapentin binding are boxed. B, phylogram. The human 2-4 subunit is 30, 32, and 61% identical to human 2-1, 2-2, and 2-3 subunits, respectively.

Genomic Structure and Splicing Variants of Human Calcium Channel ₂-4 Subunit. The full-length cDNA sequence of the human calcium channel ₂-4 subunit was used for a blast search of the GenBank human genome database. The result revealed that the gene CACNA2D4 encoding the human ₂-4 subunit is localized at chromosome 12p13.3, about 400 kb away from the locus of CACNA1C the gene of the human L-type calcium channel Ca_V1.2 (_1C) subunit (Soldatov, 1994). As shown in Fig. 2 and Table 1, the gene encoding the human calcium channel ₂-4 subunit is composed of 36 invariant exons (exon 2-exon 37) and 4 alternative exons (exon 1, 1B, 37L and 38) spanning about 130 kb of the human genome. Exon 37L is continuously extended from exon 37 with an in-frame stop codon. The genomic sequence confirms the RACE-PCR results in regard to alternative splicing. The human calcium channel ₂-4a subunit is encoded by exon 1 through exon 38, while human ₂-4b is encoded with the alternative exon 1B and exon 2 to 38. Two additional putative splicing variants are ₂-4c (exons 1-36 and exon 37L) and ₂-4d (exon 1B and exons 2-37L).

View this table: [in this window] [in a new window]   TABLE 1 Gene structure of human 2-4 subunit In the Location column, upper and lower numbers indicate the beginning and ending site of each exon on the cDNA and the deduced peptide sequences, respectively.

Tissue Distribution of Human ₂-4 Subunit. Northern blot analysis using a human ₂-4 N-terminal specific probe (1-810 bp) showed that the human ₂-4 transcript is about 7 kb and is most abundant in human heart and skeletal muscle (Fig. 4). The message level was very low in other tissues including brain, placenta, lung, liver, kidney and pancreas. The expression pattern of ₂-4 was different from that of human ₂-3, which was predominantly expressed in brain (Klugbauer et al., 1999).

View larger version (49K): [in this window] [in a new window]   Fig. 4.   Northern blot analysis of human 2-4 subunit gene expression. A, human multiple tissue blot (BD Clontech) hybridized with radioactively labeled human 2-4 subunit probe. B, same blot rehybridyzed with human 2.4-kb -actin probe (provided by BD Clontech) after stripping 2-4 subunit probe with 0.5% SDS at 90 °C for 15 min. Lane 1, heart; lane 2, brain; lane 3, placenta; lane 4, lung; lane 5, liver; lane 6, skeletal muscle; lane 7, kidney; and lane 8, pancreas.

View larger version (26K): [in this window] [in a new window]   Fig. 5.   Characterization of anti-2-3 and anti-2-4 subunit polyclonal antibodies. A to C, characterization of antibodies. SDS -PAGE and autoradiograph analysis of four different types of in vitro translated 2 subunit (A), Western blot analysis of in vitro translated 2 subunits with anti-2-3 (B), and anti-2-4 (C) antibodies. D, Western blot analysis of 2-4 subunit expression in selected human tissues. The blot was purchased from ResGen (Invitrogen). Total proteins (100 µg) from each tissue were subjected to 3 to 8% Tris-acetate gel. Lane 1, brain; lane 2, pituitary; lane 3, adrenal gland; lane 4, pancreas; and lane 5, heart.

View larger version (141K): [in this window] [in a new window]   Fig. 6.   Immunohistochemical analysis of 2-4 subunit expression in human tissues. A, Paneth cells (arrowheads) of the small intestine. B, no 2-4 immunolabeling was detected in the preabsorption control of the small intestine. Also, 2-4 immunolabeling was detected in the fetal liver erythroblasts (arrowheads) (C), in the cells of the zona reticularis (arrowheads) of the adrenal gland (D), and in the adrenal gland basophiles (arrowheads) of the pituitary (E). No 2-4 immunolabeling was detected in the tonsil (F) and cerebellum (G). Bar, 75 µm (A, B), 25 µm (D, F, G), or 100 µm (C, E).

Formation of Protein Complex in HEK293 Cells. Because the calcium channel is a multisubunit protein complex, a functional ₂-4 subunit, like other known ₂ subunits, would be a component of the complex. Formation of a stable complex of ₂-4 with other channel subunits was tested by coimmunoprecipitation. The ₂-4a subunit was first tagged with an HA epitope and cotransfected with Ca_V1.2 and ₃ subunits into HEK cells. The cell lysates were immunoprecipitated by either immobilized anti-HA monoclonal Ab or anti-Ca_V1.2 polyclonal Ab/Protein-A bead. The coprecipitated subunits were then analyzed by Western blot. Figure 7 shows that ₂-4a associated with Ca_V1.2 and ₃ after cotransfection in HEK293 cells. The expression of all three subunits in HEK293 cells was confirmed by Western blot (Figure 7A). In addition, ₂-4a(HA) was detected by both anti-₂-4 and anti-HA Abs. In Figure 7B, ₂-4a(HA) was immunoprecipitated from lysates with anti-HA Ab, and the presence of Ca_V1.2 (lane 2), ₂-4a (lane 4), and ₃ (lane 6) in the precipitates were tested by Western blot with anti-Ca_V1.2, ₂-4 and ₃ polyclonal Abs. Under the same condition, no subunits were immunoprecipitated from the cells transfected by only vector, pcDNA3.1, (Fig. 7B, lanes 1, 3, and 5). A reciprocal immunoprecipitation of Ca_V1.2 subunit from the same lysates was performed by using anti-Ca_V1.2 Ab/protein-A beads, and the presence of ₂-4(HA) was determined by Western blot with anti-HA Ab (Fig. 7C). The specific co-immunoprecipitation of ₂-4(HA) subunit was further confirmed by a negative control (Fig. 7C, lane 1), in which only protein-A beads were added.

View larger version (50K): [in this window] [in a new window]   Fig. 7.   2-4 subunit associates with CaV1.2 and 3 subunits in HEK293 cells. Reciprocal co-immunoprecipitation of CaV1.2, 3, and 2-4 (HA tagged) subunits. The HEK392 cells were transiently cotransfected by mammalian expression construct of CaV1.2, 3, and 2-4-HA. Two days after transfection, the cells were lysed and expressed, and subunits were analyzed by Western blot (A). The cell lysates were then immunoprecipitated with either anti-HA monoclonal antibody (B) or anti-1C polyclonal antibody (C), and immunoprecipitates were analyzed by Western blots with indicated antibodies.

Augmentation of Ca_V1.2/₃-Mediated Ca²⁺ Influx in HEK293 Cells. One remarkable role of the ₂ subunit is to increase levels of functional channel on the plasma membrane and thereby greatly enhance ionic current in the presence of the second regulatory subunit, the  subunit. To test whether ₂-4 subunit plays a similar role, we compared the Ca_V1.2/₃ mediated Ca²⁺ influx in the presence and absence of the ₂-4a subunit. The transfected HEK293 cells were loaded with Calcium Plus dye and depolarized by adding KCl to a final concentration of 50 mM. The changes in fluorescence intensity were measured with the Attofluor RatioVision. For analysis of the data, the fluorescence intensity changes were averaged from each individual cell regardless of whether they were expressing all the inputting subunits. However, we did exclude the cells lacking any KCl response, because vector-only transfected cells showed very little response to KCl activation (data not shown). As shown in Figure 8, upon depolarization, the Ca²⁺ influx in transfected cells was mediated by a Nifedipine sensitive Ca²⁺ channel, presumably by over-expressed Ca_V1.2 L-type Ca²⁺ channel. The Ca²⁺ influx in Ca_V1.2 transfected cells was significantly increased (~3-fold) by co-transfection of ₃ subunit. The effect was further increased to 6-fold in the presence of both ₃ and ₂-4 subunits. This result suggests that ₂-4 subunit may play a similar basic role in formation a functional channel.

View larger version (24K): [in this window] [in a new window]   Fig. 8.   The 2-4 subunit increase CaV1.2/3 mediated Ca2+ influx in HEK293 cells. Transfected cells by different combination of expression constructs as indicated were loaded with Calcium Plus dye for 1 h at 37°C. The cells were then, depolarized by adding 50 mM KCl and the intracellular [Ca2+] changes were measured in individual cells by fluorescence videomicroscopy using the Attofluor Digital Imaging system. The [Ca2+] in extracellular medium was 1.6 mM. To block Ca2+ influx, 10 µM of nifedipine was added before the depolarization. A, time course of average fluorescence intensity changes after depolarization. B, statistic analysis of changes of peak fluorescence intensity after depolarization. Each bar graph represented the means ± S.E.M. of peak fluorescence changes regardless whether all the subunits were expressed in an individual cell but excluded the cells without KCl response. The number of cells was indicated on each bar graph.

Gabapentin Binding. Gabapentin is a novel anticonvulsant drug that is also used clinically for treatment of neuropathic pain. A high-affinity gabapentin-binding site was found in brain and skeletal muscle, subsequently identified as the calcium channel ₂-1 subunit (Gee et al., 1996). More recently, Marais et al. (2001) demonstrated that gabapentin also interacted with the calcium channel ₂-2 subunit but not with the ₂-3 subunit. To determine whether gabapentin bound to the human ₂-4 subunit, we assessed specific gabapentin binding of three different types of ₂ subunit. The ₂-1, ₂-3, and ₂-4 subunits were first overexpressed in COS-7 cells by transient transfection. The membrane fractions were made 48 h after transfection and incubated with [³H]gabapentin at room temperature for 1 h. The membranes were subsequently filtered, and gabapentin binding to the membrane was quantified by scintillation counting. As shown in Fig. 9, gabapentin bound to the ₂-1 subunit with a relatively high affinity, but it failed to bind to either the ₂-3 or ₂-4 subunits. This result is consistent with Marais' observation (Marais et al. 2001) and further supports the gabapentin binding pharmacophore characterized by Wang et al. (1999), which is present in the ₂-1 and ₂-2 but not in the ₂-3 and ₂-4 subunits.

View larger version (30K): [in this window] [in a new window]   Fig. 9.   Gabapentin does not bind to 2-4. COS-7 cells were transiently transfected 2 subunit expression constructs and parental vector, pcDNA3.1, separately. After 48 h, transfected cells were collected and membrane fraction was made. A, Western blot analysis of membrane fractions. Membrane protein (50 µg) from each sample was subjected to 4 to 12% SDS-PAGE and transferred to nitrocellulose after electrophoresis. The blots were probed with anit-2-1, -3, and -4 polyclonal antibodies, respectively. Lanes 1, 3, and 5, cells transfected by pcDNA3.1; lane 2, by 2-1/pcDNA3.1; lane 4, transfected by 2-3/pcDNA3.1; and lane 6, by 2-4/pcDNA3.1. B, [3H]gabapentin binding to the membrane fraction of transfected cells. The assay was performed at room temperature with 40 µg of total membrane protein from each sample and 20 nM [3H]gabapentin in a final volume of 200 µl. Values are the means ± S.D. of numbers of experiments indicated on top of each bar.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

We report here the molecular cloning and characterization of a novel human voltage-gated calcium channel ₂-4 subunit and its amino and carboxyl terminal splicing variants. We also characterized its gene structure and determined its tissue distribution pattern by Northern blot analysis and immunohistochemistry. We conclude that this novel protein is a new member of the ₂ subunit family based on the following observations. First of all, the ₂-4 subunit shares 30, 32, and 61% identity with human calcium channel ₂-1, ₂-2, and ₂-3 subunits, respectively. Secondly, the ₂-4 subunit possesses most of the known ₂ subunits molecular features. These include a potential signal sequence at the amino terminus, a transmembrane domain at the carboxyl terminus, 15 conserved cysteine residues, multiple glycosylation sites, and conserved regions clustered on the ₂ portion. Finally, when the ₂-4 subunit was expressed in HEK293 cells, it formed a protein complex with Ca_V1.2 and ₃ subunits (Fig. 7), and increased Ca²⁺ influx mediated by Ca_V1.2/₃ channel (Fig. 8). Comparison of the primary sequences of all the ₂ subunits (Fig. 3) also showed that the ₂-1 and ₂-2 subunits belong to one subfamily, whereas the ₂-3 and ₂-4 subunits belong to another subfamily. Interestingly, although the ₂-4 subunit is most similar to the ₂-3 subunit in primary sequence, its tissue distribution pattern is quite different. The reported protein distribution of the ₂-3 is predominately in brain tissue (Klugbauer et al., 1999), whereas the ₂-4 subunit is in certain types of endocrine cells, suggesting that ₂-3 and ₂-4 may play regulatory roles in different tissues.

Like the ₂-2 subunit, one of the N-terminal isoforms of the ₂-4 subunit (₂-4a) consists of a potential signal sequence, whereas another (₂-4b) does not. The isoform without the potential signal peptide may not form a typical ₂ subunit membrane topology and will not be highly glycosylated, which is believed to be critical for the regulatory activity of ₂ subunits. However, the existence and biological function of the ₂ subunit isoforms without a putative signal peptide is still not understood. It will be interesting to see whether the isoform with such different membrane topology plays a different role in the regulation of calcium channel activity.

The ₂-1 subunit is known to increase the number of functional channels on the cell surface and to alter the binding of neurological and cardiovascular drugs to the ion channel pore-forming ₁ subunit. Recently, it has been shown that the calcium channel ₂ subunit contains a binding site for gabapentin (Gee et al. 1996). Gabapentin may control neuronal excitability by modifying calcium channel activity (Rock et al. 1993) providing its therapeutic role in epilepsy and neuropathic pain. Recently, Wang et al. (1999) has identified four regions on ₂ subunit that are required for gabapentin binding. The similar binding motif may exist in the ₂-2 subunit, but not in the ₂-3 and the novel ₂-4 subunits. In support of this notion, we found that ₂-3 and ₂-4 subunits do not bind to gabapentin. Obviously, gabapentin might be a less safe medication if it bound to all subtypes of ₂ subunits and subsequently altered the activity of all calcium channels. The finding that gabapentin binds neither to the ₂-4 subunit (present study) nor to the ₂-3 subunit (Marais et al., 2001) may partially explain why gabapentin has certain clinical properties or alters only certain types of calcium channel activities. At this point, we cannot exclude the possibility that the effect of gabapentin may also depend on the specific types of ₁ and  subunits that are associated with the gabapentin-binding ₂ subunits (₂-1 and ₂-2).

Because the gene encoding the ₂-4 subunit is colocalized with the _1C subunit (Ca_V1.2) on human chromosome 12p13.3, with a distance of only about 400 kb, an obvious question would be whether these two subunits associate to form a functional channel in native tissue. It is well known that the major subtypes of Ca_V1.2 (_1C) are predominantly expressed in heart, smooth muscle and brain, a distribution significantly different from that of the ₂-4 subunit reported here (Fig. 6) in the pituitary, adrenal gland, small intestines, and fetal liver tissue. This suggests that the ₂-4 subunit is not a component of L-type channel in heart, brain, and smooth muscle. Interestingly, the Ca_V1.2c (_1C-C, or rbC-I/rbc-II), one of the splicing variants of the _1C subunit, is also expressed in pituitary and adrenal gland (Snutch et al., 1991), a localization consistent with that of the ₂-4 subunit. In addition, several other types of ₁ subunits, such as Ca_V1.3, Ca_V2.1, and Ca_V2.3 are also expressed in pituitary. Further study will be necessary to elaborate the physiological role of calcium channel ₂-4 subunit. The limited expression pattern of the ₂-4 subunit in normal human tissues also includes possible roles in the processes of erythrogenesis as the erythroblasts loose their nuclei. Also, the presence of the ₂-4 subunit in the cells of the pituitary and adrenal glands and in the Paneth cells of the small intestine suggests its roles in calcium-mediated exocytosis.

While this article was in preparation, a partial cDNA sequence (GenBank accession number XM_052387) encoding carboxyl terminal 378 residues of human ₂-4 subunit was deposited by the National Center for Biotechnology Information, National Institutes of Health. It was annotated as "Homo sapiens similar to voltage-dependent calcium channel mouse ₂-3 subunit."