Cloning of a functional splice variant of L-type calcium channel beta 2 subunit from rat heart.

L-type Ca(2+) channels are heteromultimeric and finely tuned by auxiliary subunits in different tissues and regions. Among auxiliary subunits, beta subunit has been shown to play important roles in many functional aspects of Ca(2+) channel. Rat heart was reported to specifically express beta(2a) subunit. However, the slow inactivation rates of Ca(2+) currents recorded from recombinant Ca(2+) channels with the beta(2a) subunit, and the reported inability to detect beta(2a) subunit in rabbit heart by reverse transcription-PCR analysis raise the possibility of the existence of other beta subunits. We cloned a splice variant of beta(2) subunit from rat heart, using rapid amplification of cDNA 5' ends. The splice variant is highly similar to human beta(2c) subunit that was cloned from human ventricle. Northern blot analysis detected the rat beta(2c) subunit abundantly in rat heart and brain. The deduced amino acid sequence of the beta(2c) subunit was different from that of the beta(2a) subunit only in the N-terminal region. When the beta(2c) subunit was expressed along with alpha(1c) and alpha(2)delta subunits in baby hamster kidney cells, the inactivation rates were comparable with those from native cardiac myocytes, although those with the beta(2a) subunit were slow. Taken together, these observations suggest that the beta(2c) subunit is a functional beta(2) subunit expressed in heart and that the short N-terminal region plays a major role in modifying inactivation kinetics.

L-type Ca 2ϩ channel plays an important role in shaping the action potential of cardiac myocytes and is a major pathway for extracellular Ca 2ϩ entry into cardiomyocytes (1). Four distinct subunits, i.e. ␣ 1c , ␤, and ␣ 2 ␦, compose the L-type Ca 2ϩ channel in tissues other than skeletal muscle (2). Among auxiliary subunits, ␤ subunit has been shown to be a central player in most, if not all, functional aspects of Ca 2ϩ channel (3). ␤ subunit promotes the transportation of Ca 2ϩ channel to the membrane and affects the activation and inactivation kinetics (3). To date, four distinct ␤ subunits, i.e. ␤ 1 , ␤ 2 , ␤ 3 , and ␤ 4 , and their splice variants have been cloned and expressed in different tissues and regions (2)(3)(4)(5)(6)(7)(8). All four ␤ subunits are expressed in brain (3)(4)(5)(7)(8)(9). In contrast, the skeletal and cardiac muscle Ca 2ϩ channels are thought to be associated, apparently exclusively, with the ␤ 1a and cardiac ␤ 2 subunits, respectively (6). Indeed, rat ␤ 2a (8), rabbit ␤ 2a (7), and ␤ 2b (7) subunits, the difference among which resides only in the N-terminal region, were reported to be respectively expressed in these animal hearts. However, in human heart, mRNA expression of ␤ 1b (3,10,11), ␤ 1c (3,10), and ␤ 3 (11) subunits has been demonstrated in addition to protein expression of the ␤ 2 subunit (12). Moreover, recent PCR analysis could not detect the rat ␤ 2a subunit with the rat ␤ 2a -specific primers in rabbit heart, but it was present in brain (13). Therefore, it is not yet clear which ␤ subunits exist in heart and whether heart expresses certain ␤ subunits specifically.
Coexpression of the rat ␤ 2a subunit with various ␣ 1 subunits slowed the inactivation of the Ca 2ϩ current (I Ca ), although all the other ␤ subunits accelerated it (5)(6)(7)14). Inactivation rates of I Ca from native cardiac Ca 2ϩ channels are apparently fast, compared with those from L-type Ca 2ϩ channels containing the rat ␤ 2a subunit in a heterologous expression system (15). This fact, by itself, implies that other ␤ subunits and/or regulators may function in rat heart. However, no evidence was found to show that the ␤ 1 , ␤ 3 , and/or ␤ 4 subunits existed in rat heart (4,5,8). These observations led us to speculate about the possible existence of unidentified ␤ subunits and/or splice variants in heart.
Indeed, rapid amplification of cDNA 5Ј ends (5Ј-RACE) 1 disclosed a ␤ 2 transcript in rat heart, which was only different from other ␤ 2 subunits in the N-terminal region. Here, we named it rat ␤ 2c subunit, because the sequence of this subunit was very similar to that of human ␤ 2c subunit, which was also cloned from human ventricular septum. When the ␤ 2c subunit was expressed in BHK cells along with a pore-forming ␣ 1c and other auxiliary ␣ 2 ␦ subunits cloned from rat heart, the inactivation rate was comparable with that from native cardiac myocytes.

EXPERIMENTAL PROCEDURES
Preparation of Single Cardiac Myocytes-Single ventricular myocytes were enzymatically isolated from the ventricle of rat hearts as described previously (16). In brief, the hearts were removed from rats, following anesthetic with pentobarbital, and perfused in a Langendorff apparatus with 0.02-0.04% collagenase (Wako Pure Chemical Industries, Osaka, Japan) dissolved in nominally Ca 2ϩ -free Tyrode solution. After 30 min of digestion, the left ventricle was rinsed with Kraftbrü he (KB) solution (17), cut into small pieces, and shaken to separate cells.
RNA Isolation and RT-PCR Analysis-Rats weighing 250 -350 g were anesthetized and killed with an excessive amount of pentobarbital, and the hearts and brains were removed as quickly as possible and frozen in liquid nitrogen. Total RNAs and messenger RNAs (mRNAs) were extracted using TRIzol ® reagent (Life Technologies, Inc.) and a FastTrack ® 2.0 kit (Invitrogen), respectively, according to the manufacturers' instructions. When we had determined whether a certain mRNA was expressed or not in heart and/or brain, RNA samples were treated with DNase I (Life Technologies, Inc.) before cDNA synthesis to eliminate contamination of genomic DNA. However, we did not use DNase when our purpose was to clone ␣ 1c , ␤ 2a , ␤ 2c , and ␣ 2 ␦ subunits that contained a full-length open reading frame (ORF). RNA was quantified by 260/280 ultraviolet (UV) spectrophotometries. Total RNAs (2 g) of rat hearts and brains were reverse transcribed for 40 min at 42°C with 200 units of Superscript™ II reverse transcriptase (Life Technologies, Inc.), using oligo(dT) primers or random hexamers. RT was performed in a 20-l mixture containing 20 mM Tris-HCl (pH8.4), 50 mM KCl, 2.5 mM MgCl 2 , 500 M amounts of each dNTP, 10 mM dithiothreitol, and 500 nM oligo(dT) or random hexamer primers. PCR was performed by using 1 l of each RT product as template DNA and primers (listed in Table I). PCR reactions (50 l volume) contained: 60 mM Tris-HCl (pH 9.1), 18 mM (NH 4 ) 2 SO 4 , 200 M amounts of each dNTP, 0.4 M each of the primers (see Table I), and 1.6 mM MgCl 2 . The ELONGASE™ enzyme mix was added to each sample after denaturation at 94°C for 3 min. Amplification was performed in a PCR Thermal Cycler MP (Takara) for 35 cycles at 94°C for 30 s, 62-64°C for 20 -30 s, and 68 -72°C for 1-7 min. Each sample (1 l) of the RT-PCR products was analyzed by electrophoresis in 0.8 -2% agarose gel and visualized by UV fluorescence, using a FluorImager SI (Molecular Dynamics, Sunnyvale, CA) after staining with ethidium bromide for 15 min or Vistra Green (Amersham Biosciences, Inc.) for 1 h. The specificity of the PCR products was confirmed by their predicted lengths on agarose gels and DNA sequence (373 DNA Sequencing System, Applied Biosystems) after they had been subcloned into pGEM ® -T Vector (Promega) or pCR ® -XL-TOPO vector (Invitrogen).
5Ј-RACE-When the ␤ 2c subunit was cloned, 5Ј-RACE was performed with 1 g of total RNA from rat heart to obtain the unknown forward primer sequence, using a 5Ј-RACE System for Rapid Amplification of cDNA Ends (Life Technologies, Inc.). The first-strand cDNA was synthesized by reverse transcription with a ␤ 2 -specific primer, ␤2primer_for_5ЈRACE604r1 (complementary to 459 -479 bp from the translation start site, Table I). 5ЈcDNA ends of ␤ 2c were amplified using nested PCR with primers, ␤2primer_for_5ЈRACE604r2 and ␤2primer_for_5ЈRACE604r3 (complementary to 395-415 and 355-375 bp relative to the translation start site, respectively, Table I). PCR products were cloned into pGEM ® -T vector, and the inserts were sequenced.
Northern Blot Analysis-Antisense riboprobes containing heart-and brain-specific N-terminal regions were constructed. Briefly, RT-PCR amplification of the ␤ 2a -and ␤ 2c -specific fragments was performed, using the primers listed in Table I. They were subcloned into pGEM ® -T vectors to form plasmids pGEM-fr␤ 2a and pGEM-fr␤ 2c . Radiolabeled antisense riboprobes were synthesized from NotI-linearized pGEM-fr␤ 2a and pGEM-fr␤ 2c with T7 RNA polymerase and [ 32 P]UTP using Strip-EZ RNA™ (Ambion) according to the manufacturer's instructions. Ten g of poly(A) RNA samples from rat heart and brain were denatured, separated on a 1% agarose gel containing 7.4% formamide, and then transferred to Immobilon-N (Millipore). Northern blots were prehybridized for 30 min at 68°C in the presence of Ultrahyb™ (Ambion). The blots were hybridized overnight at 68°C in the same solution with the 10 6 cpm/ml 32 P-labeled riboprobes described above. The membrane was then washed at 68°C with 2ϫ SCC plus 0.1% SDS twice for 5 min and with 0.1ϫ SCC plus 0.1% SDS twice for 15 min, and then autoradiographed for 7 days at Ϫ20°C.
Electrophysiological Study-Coverslip fragments with attached cells or isolated cardiac myocytes were continually perfused on the stage of an inverted microscope. Whole-cell patch-clamp recordings were made using pipettes with resistance of 2-4 megohms. Perfusate composition was as follows (in mM): choline chloride 140, CsCl 5.4, MgCl 2 0.5, Reverse primer for a full-length ␤ 2c GCAATGGAGCCCCAAAGATT HEPES 5, and D-glucose 10 (pH 7.4, titrated by Tris-HCl), with Ba 2ϩ 10, 0.5, or Ca 2ϩ 1.8 as charge carrier. Pipette solution composition was as follows (in mM): l-glutamic acid 110, CsCl 20, CsOH 110, MgCl 2 1, Na 2 ATP 5, creatine phosphate 5, EGTA 10, and HEPES 5 (pH ϭ 7.4, titrated by 1 N CsOH). We chose BHK and COS-7 cells as heterologous expression systems because they were reported to lack any subunits of L-type Ca 2ϩ channel (20 -22). All experiments were done at 37°C. Transfected cells were identified by the expression of hrGFP. Cells were clamped at Ϫ80 mV, and whole-cell currents were evoked by 400-ms step depolarization to various test potentials (0.1Hz). Currents were filtered at 2 kHz and digitized at 10 kHz. Series resistance compensation was not applied. Analysis and voltage protocols were performed with the use of an Axopatch 1D amplifier/Digidata 1322A interface (Clampex software, pCLAMP 8.1, Axon Instruments Inc). The data were analyzed after leak subtraction. Inactivation characteristics of Ba 2ϩ current (I Ba ) were measured using a two-step voltage clamp protocol. A 3-s conditioning pre-pulse was applied from a holding potential of Ϫ80 mV (10-mV increments from Ϫ100 mV to 10 mV) followed by a 100-ms test pulse to 10 mV. The intervals between each cycle were 10 s. Recorded peak current amplitudes were normalized to the maximum value. Steady-state inactivation curves were fitted by a Bolzmann function: where I Ba is the normalized peak current, V is the conditioning pre-pulse voltage, V h is the voltage at half-maximum inactivation, and k is the slope factor. Inactivation time constants were obtained by fitting a current decay with a two-exponential equation: the time-dependent current, I ϱ is the steady-state amplitude of the current, A s and A f are the fractions of currents that are slow and fast inactivated, respectively, and slow and fast are the time constants that are slow and fast, respectively. The r400 value was calculated as the ratio of the current amplitude at the end of 400-ms depolarization pulse divided by the peak amplitude of the trace. Statistics-All values are presented in terms of mean Ϯ S.E. When I-V relationships and inactivation characteristics were compared, twoway repeated measures of ANOVA were first carried out to test for any differences among the mean values of multiple subgroups. When a significant F value was obtained by two-way ANOVA, intergroup comparisons were performed by contrast test. When the model with the interactions improved significantly, we interpreted it as indicating different voltage dependences of the groups. The voltages at half-maximum inactivation and the slope factors were compared by Student's t test. Significance was established at P values Ͻ 0.05.

RESULTS
First, we performed RT-PCR to confirm that the ␤ 2 subunit exists in rat heart, using total RNA extracted from rat heart as a template. As shown in lane 1 of Fig. 1B, we were able to obtain a partial fragment of ␤ 2 subunit. Then, we attempted to isolate the reported ␤ 2a subunit including a full-length ORF. However, as shown in lane 2 of Fig. 1B, we could not observe any PCR product using the ␤ 2a -specific primers. As the same primers detected the ␤ 2a subunit when total RNA from rat brain (Fig. 1B, lane 5) was used as a template, we speculated that another splice variant of ␤ 2 was expressed in rat heart. The ␤ 2 subunit is known to have splice variants in the Nterminal region. To prove that the forward primer was not suitable to obtain the ␤ 2 subunit in rat heart, we performed RT-PCR analysis using a primer pair of primer_1 and primer_4 and another pair, primer_3 and primer_2 (see Fig. 1A). As shown in lanes 3 and 4 of Fig. 1B, only primer_3 and primer_2 detected the ␤ 2 subunit, which indicated that the N-terminal region of the ␤ 2 subunit expressed in rat heart was different from the reported ␤ 2a sequence cloned from rat brain.
The upper lane of Fig. 2A shows the partial sequence of the ␤ 2c subunit in rat heart that was determined by 5Ј-RACE (GenBank™ accession number AF394942). The sequence was completely different from the reported ␤ 2a sequence in the 5Ј-untranslated region (UTR) and the first 199 nucleotides of ORF. We set the primers based on this result and succeeded in cloning the full-length ␤ 2c subunit (GenBank™ accession number AF394941) expressed in rat heart (Fig. 3A, lane 2). This ␤ 2c clone contains 656 amino acids, yielding a protein with a calculated molecular mass of 73.2 kDa. We could also amplify the ␤ 2c subunit in rat brain, using the same primers (Fig. 3A, lane  1). Therefore, it was demonstrated that the ␤ 2c subunits were expressed in heart and brain.
The deduced partial amino acid sequences for the rat ␤ 2c and ␤ 2a subunits are compared in Fig. 2B, along with that for the human ␤ 2c , the rabbit ␤ 2a , and the rabbit ␤ 2b subunits (7,8,23). The rat ␤ 2c subunit differs only in the N-terminal region from the rat ␤ 2a , the rabbit ␤ 2a , and ␤ 2b subunits except for some amino acids sequence, considered to be species differences. The N-terminal region of the rat ␤ 2c subunit has one potential protein kinase C site and two casein kinase II phosphorylation sites. No consensus site for cAMP-dependent protein kinase was found in this region. In contrast to other ␤ 2 subunits, the sequence for the N-terminal region of the human ␤ 2c subunit, which was also cloned from human ventricular septum (23), is almost identical to that for the rat ␤ 2c subunit.
To confirm the PCR result further, Northern blot analysis was performed. The ␤ 2c -specific probe hybridized with three mRNA species of ϳ6, 4, and 2 kb in rat heart (Fig. 3B, lane 3). However, the ␤ 2a -specific probe hybridized only two transcripts of about 6 and 2 kb (Fig. 3B, lane 1). In rat brain, both the ␤ 2aand the ␤ 2c -specific probes cross-reacted with three transcripts of ϳ6, 4, and 2 kb (Fig. 3B, lanes 2 and 4). Perez-Reyes et al. (8) detected mRNA species of 6, 4, and 3.5 kb in rat heart, using 32 P-labeled randomly primed cDNA of the ␤ 2 clone. As the sequence of the ␤ 2a and the ␤ 2c subunits are the same except for the N-terminal region, it is highly possible that the probe used by Perez-Reyes et al. (8) contained the common sequences that recognize both the ␤ 2a and the ␤ 2c subunits. Therefore, the 4-kb transcript, which was not recognized by the ␤ 2a -specific probe but by the probe of Perez-Reyes et al., may be the ␤ 2c subunit. These results, along with the PCR analysis, demonstrated that the ␤ 2c subunit was expressed abundantly in both heart and brain (see also "Discussion").
To test the functional effect of the ␤ 2c subunit and to compare the recombinant Ca 2ϩ channel containing the ␤ 2c subunit with native one, we cloned a pore-forming subunit and other auxil- The primer_1, primer_2, primer_3, and primer_4 correspond to total-beta2-primer414f, total-beta2-primer414r, ␤2primer302f, and ␤2primer302r, respectively, in Table I iary subunits of L-type Ca 2ϩ channel, i.e. ␣ 1c and ␣ 2 ␦ subunits from rat heart. We thought it important to reconstitute the L-type Ca 2ϩ channel, which was composed of all four subunits cloned from heart of the same species. Otherwise, any different characteristics from native channels may have been the result of subunits other than those of ␤ subunits. We obtained three kinds of splice variants of ␣ 1c subunit (i.e. ␣ 1c5 , ␣ 1c12 , and ␣ 1c15 ; GenBank™ accession numbers AF394938, AF394939, and AF394940, respectively). The sequence of the cloned ␣ 1c subunits consists of an ORF of 6513 bases that encodes 2171 amino acids. The calculated molecular masses are about 243 kDa. They differ only at two splice regions (Fig. 4) (2). The ␣ 1c15 displays a 99% amino acid identity with the rat aortic ␣ 1c and a 97% identity with the rat neuronal ␣ 1c (18,24). The ␣ 1c15 and the rat aortic ␣ 1c are mainly different only at the splice region IIIS2, whereas the ␣ 1c15 and the rat neuronal ␣ 1c are only different at the splice region's N terminus and IVS3_S3-S4(2). As we set the forward primer in 5Ј-UTR and the reverse primer in 3Ј-UTR, the sequences obtained were actually considered to exist as mRNAs. It is of note that the reported specific splicing site for heart and smooth muscle of the clones obtained at this time was identical to that of the heart, although the sequence of the site of ␣ 1c15 was exactly the same as that of the reported rat aortic ␣ 1c (25). We chose ␣ 1c15 because it was the easiest clone with which to correct the PCR error. As for ␣ 2 ␦ subunit, the nucleotide sequence of our clone (GenBank™ accession number AF400662) was the same as that of the reported ␣ 2 ␦, which is one of the ␣ 2 ␦ splice variants expressed in mouse heart (26) (i.e. ␣2d, GenBank™ accession number AAB50141) except for some of the sequence, which are considered to be species differences. Angelotti et al. (26) reported that mouse heart was found to express ␣2c and ␣2d in approximately a 9:1 ratio, respectively. We had four clones that contained a full-length ␣ 2 ␦ subunit. However, all of them corresponded with the mouse ␣2d subunit.
When an ion channel protein is expressed in a heterologous expression system and the patch-clamp experiment is done, GFP is often attached to the N terminus or C terminus of the channel protein, to choose the transfected cell easily. However, it has recently become known that this attached GFP changes the kinetics of the channel itself (15). To avoid these problems, we used pIRES that contained a pore-forming ␣ 1c subunit along with one of the ␤ 2 subunits. Besides this, we transfected cells with pIRES-hrGFP or pIRES-␣ 2 ␦-hrGFP simultaneously. These vectors contain the internal ribosome entry site (IRES) from the encephalomyocarditis virus, which allows translation of two consecutive open reading frames from the same mRNA (27). In this case, the single mRNA species supports translation of separate ␣ 1c and one of the ␤ 2 proteins or ␣ 2 ␦ and hrGFP proteins. Thus, if we could record I Ba from a cell expressing hrGFP, it was highly possible that recombinant Ca 2ϩ channels contained ␣ 1c and one of the ␤ 2 subunits (along with the ␣ 2 ␦ subunit when pIRES-␣ 2 ␦-hrGFP was used.). Fig. 5 (A and B) shows the representative traces of I Ba that we recorded from BHK cells transfected with pIRES-␣ 1c -␤ 2a and pIRES-␣ 1c -␤ 2c , respectively. Ca 2ϩ channels were expressed in about 70 -90% of the hrGFP-expressing cells. We used 10 mM Ba 2ϩ as charge carrier to circumvent the effect of Ca 2ϩ on inactivation kinetics and to make the difference in voltage-dependent inactivation kinetics clearer. I Ba was blocked by the dihydropyridine derivative, nifedipine (10 M, data not shown). We judged the ␤ 2 subunit to have functioned if the current was FIG. 2. A, nucleotide sequence comparison between ␤ 2c and ␤ 2a subunits. 5Ј-RACE analysis of rat heart was performed as described under "Experimental Procedures," and the sequence of ␤ 2c subunit was determined. Upper and lower lanes indicate the sequence of ␤ 2c and ␤ 2a subunits, respectively. A site of sequence identity is marked with an asterisk. The sequence of the ␤ 2a subunit was from Perez-Reyes et al. (GenBank™ accession number M80545). The nucleotide sequence of the rat ␤ 2c subunit can be retrieved using GenBank™ accession number AF394942. B, Alignment of the deduced amino acid sequence of rat ␤ 2c subunit. Amino acid sequences of rat ␤ 2c , rat ␤ 2a , human ␤ 2c (GenBank™ accession number AF137376), rabbit ␤ 2a (Gen-Bank™ accession number X64297), and rabbit ␤ 2b (GenBank™ accession number X64298) are aligned for comparison. The potential sites for protein kinase C (f) and casein kinase II (E) phosphorylation are indicated above the line. Identical amino acids are indicated by vertical bars. Dashes represent gaps in the sequence. larger than 100 pA, because recombinant Ca 2ϩ channel currents expressed without ␤ subunit were very small (typically 10 -50 pA, data not shown), as reported elsewhere (21,22). It is apparent that the current with ␤ 2c inactivated faster than that with ␤ 2a . The fractions of current remaining at the end of 400-ms depolarization (r400) derived from traces of ␤ 2c -trans-fected cells were significantly smaller throughout the membrane potentials examined than those with ␤ 2a , though I-V relations were not different (Fig. 5, C-E). The steady-state inactivation curves derived from records with ␤ 2a and ␤ 2c were not significantly different (Fig. 5F). The rates of inactivation with ␤ 2c were apparently faster than those with ␤ 2a even when we used COS-7 cells instead of BHK cells (data not shown). The r400 values derived from COS-7 cells with ␤ 2c and ␤ 2a showed the same tendency as those from BHK cells (Fig. 5E). When pIRES-␣ 2 ␦-hrGFP was transfected to BHK cells in addition to pIRES-␣ 1c -␤ 2a or pIRES-␣ 1c -␤ 2c , I Ba with ␤ 2c transfection also inactivated faster than I Ba with ␤ 2a transfection (Fig. 6, A and  B; charge carrier was 0.5 mM Ba 2ϩ (see the reason below)). The same observation in different expression systems with or without other auxiliary subunits strongly suggests that the ␤ 2a and the ␤ 2c subunits function differently.
To compare the inactivation rates from recombinant Ca 2ϩ channels with those from native cardiac myocytes, the concentration of charge carrier Ba 2ϩ ion that flows through Ca 2ϩ channel was changed from 10 to 0.5 mM. Otherwise, the peak amplitudes of I Ba recorded from native cardiac myocytes was FIG. 3. A, RT-PCR detection of a full-length ␤ 2c subunit. RT-PCR was performed using tbeta2-p-308f as the forward and tbeta2-p-308r as the reverse primers (see also Table I). PCR products with an expected length of ϳ 2 kb were detected from adult rat brain (lane 1) and adult rat heart (lane 2). B, Northern blot analysis of ␤ 2a and ␤ 2c subunits. Northern blots of mRNA from adult rat brain (lanes 2 and 4) and heart (lanes 1 and 3) were hybridized with probes specific for ␤ 2a (lanes 1 and  2) and ␤ 2c (lanes 3 and 4), as described under "Experimental Procedures." Closed triangles indicate the 2-, 4-, and 6-kb transcripts hybridized with the probes. Note there is no 4-kb transcript in lane 1.

FIG. 4.
Comparison of three splice regions of ␣ 1c subunit. The deduced amino sequences of ␣ 1c subunits cloned from rat heart (␣ 1c15 , ␣ 1c5 , and ␣ 1c12 ), rat aorta (GenBank™ accession number M34364) and rat brain (rbC-l and rbC-ll, GenBank™ accession numbers M67515 and M67516) are aligned for comparison of the three splice regions. Dashes represent identical amino acid sequences. The nucleotide sequence of ␣ 1c5 , ␣ 1c12 , and ␣ 1c15 can be retrieved using the GenBank™ accession numbers AF394938, AF394939, and AF394940, respectively. Lower panel, comparison of the r400 values (E) and steady-state inactivation curves (F) between the ␤ 2a and ␤ 2c subunits. The r400 values of I Ba with ␤ 2a and ␤ 2c were calculated as described under "Experimental Procedures." Closed squares and circles represent data from BHK cells transfected with ␤ 2a (f, n ϭ 10) and ␤ 2c (q, n ϭ 12). Open squares and circles represent data from COS-7 cells transfected with ␤ 2a (Ⅺ, n ϭ 8) and ␤ 2c (E, n ϭ 10). Asterisks in panel E show statistical significance by two-way repeated measures of ANOVA. Steady-state inactivation curves (F), which were derived from I Ba with the ␤ 2a (f, n ϭ 8) and ␤ 2c (q, n ϭ 10) subunits expressed in BHK cells, were not significantly different (two-way repeated measures of ANOVA). The V h values with ␤ 2a and ␤ 2c were Ϫ39.2 Ϯ 1.77 mV and Ϫ36.4 Ϯ 1.59 mV, respectively (not significant by t test). The k values with ␤ 2a and ␤ 2c were 9.08 Ϯ 1.04 and 7.96 Ϯ 0.43, respectively (not significant by t test). Charge carrier was 10 mM Ba 2ϩ ion. over 10 nA and it became difficult to voltage-clamp a cell. As seen in the representative traces (Fig. 6, upper traces), the inactivation rates from a native cardiac myocyte were very similar to those from a BHK cell with ␤ 2c , but different from those with ␤ 2a . A current decay was fitted to a two-exponential function significantly better than a single-exponential function. Fig. 7 shows comparisons of time constants ( fast and slow ) and their contribution (A s /A f ) calculated using traces from BHK cells with ␤ 2a , ␤ 2c , and native cardiac myocytes. slow (Fig. 7A), fast (Fig. 7B), and A s /A f (Fig. 7C) obtained from native cardiac myocytes were significantly smaller than those from BHK cells with ␤ 2a (statistically significant by ANOVA and contrast test), whereas they were very similar to those with ␤ 2c (not significant by contrast test). However, we could not reconstitute Ca 2ϩ channel expressed in native heart completely. The r400 values of native I Ba were smaller than those of ␤ 2a , as expected, but they were larger than those of ␤ 2c (Fig. 7D). Besides, the voltage dependence of the normalized I-V relationship from native cardiac myocytes was slightly but significantly different from that from BHK cells with ␤ 2a or ␤ 2c subunit (Fig. 6, D-F). However, these observations strongly suggest that the ␤ 2c subunit functions in cardiac myocytes.
To examine the inactivation kinetics in more physiological conditions, we changed the charge carrier from 0.5 mM Ba 2ϩ to 1.8 mM Ca 2ϩ . The upper panel of Fig. 8 shows representative traces recorded from BHK cells expressing recombinant channels and a native cardiac myocyte. Ca 2ϩ accelerated current decays in all traces compared with those recorded in the solution containing Ba 2ϩ . However, I Ca through the recombinant Ca 2ϩ channel with ␤ 2a (Fig. 8A) was, apparently, still slower to inactivate than with ␤ 2c (Fig. 8B) and the native cardiac channel (Fig. 8C). In contrast, it was difficult to distinguish between the traces recorded from the BHK cells transfected with ␤ 2c (Fig. 8B) and the native cardiac myocyte (Fig. 8C). When a decay of I Ca was fitted to a two-exponential function, fast (Fig.  8H) obtained from BHK cells with ␤ 2a was significantly slower than that from BHK cells with ␤ 2c and native cardiac myocytes, and fast from recombinant channels containing ␤ 2c and native channels were not different statistically as expected. However, slow (Fig. 8G) and A s /A f (Fig. 8I) calculated from all three groups were significantly different from each other (ANOVA and contrast test). Moreover, two-way repeated measures of ANOVA and contrast test detected significant difference in the voltage dependence of I-V relations among all three groups (Fig. 8, D-F). These observations also suggest that some Ca 2ϩdependent modulators are needed to reconstruct Ca 2ϩ channel, because the inactivation kinetics of I Ba derived from recombinant channels containing ␤ 2c and native cardiac channels were similar but those of I Ca were somewhat different. However, the similarity of the inactivation kinetics obtained using ␤ 2c to those of the native ones, compared with those from ␤ 2a , supports our hypothesis that ␤ 2c subunit functions in heart. DISCUSSION In rat heart, the ␤ 2a subunit was reported to be expressed in a tissue-specific manner (8). However, recent reports cast doubt on the functional roles of ␤ 2a subunit in cardiac myocytes. Wei et al. (15) proposed the possibility that other ␤ subunits function dominantly, because they could not reconstitute similar inactivation rates of I Ba , even when cardiac myocytes were transfected with the ␤ 2a subunit. Qin et al. (13) reported that PCR analysis could not detect any signal in rabbit heart with the rat ␤ 2a -specific primers. In an attempt to study cardiac Ca 2ϩ channel using the RT-PCR method, we also failed to obtain the ␤ 2a subunit from rat heart with the rat ␤ 2a -specific primers, but obtained it from brain. Historically, the rat ␤ 2a subunit was first cloned from brain (8); then, Northern blot analysis demonstrated that this subunit was also expressed in heart (8). We were able to obtain a partial fragment of ␤ 2a subunit from heart. However, with the specific primer for the N-terminal region of the ␤ 2a subunit, although we were able to obtain a signal from brain, no signal could be procured from heart. From these observations, we believed that rat heart did not express the N-terminal region of the ␤ 2a subunit. Therefore, we were obliged to perform 5Ј-RACE. As a result, we cloned a splice variant of ␤ 2 subunit, called here the rat ␤ 2c subunit. Northern blot analysis demonstrated that the ␤ 2c subunit was expressed abundantly in heart and that a part of the ␤ 2a subunit considered to be expressed in heart might be, in fact, FIG. 6. Comparison between recombinant and native Ca 2ϩ channels. Charge carrier was 0.5 mM Ba 2ϩ ion to compare I Ba through recombinant and native Ca 2ϩ channels in the same environment. To reconstruct Ca 2ϩ channels in BHK cells, the ␣ 2 ␦ subunit was added along with ␣ 1c and one of the ␤ 2 subunits. The upper panel shows representative I Ba traces from BHK cells transfected with ␤ 2a (A), ␤ 2c (B), and a native cardiac myocyte (C). Vertical bar and horizontal bar indicate 500 pA and 50 ms, respectively. Lower panel shows I-V relationships derived from BHK cells transfected with ␤ 2a (D, n ϭ 5), ␤ 2c (E, n ϭ 5) subunits, and native cardiac myocytes (F, n ϭ 5). Currents were normalized by peak current amplitude. Two-way repeated measures of ANOVA found significant difference in voltage dependence among the three groups. Contrast test indicated that voltage dependences were significantly different between ␤ 2a and native groups, and between ␤ 2c and native groups.  2a (f (A, B, and D), open column (C), n ‫؍‬ 5), ␤ 2c (q (A, B, and D), closed column (C), n ‫؍‬ 5) and native cardiac myocytes (‚ (A, B, and D), hatched column (C), n ‫؍‬ 5). A current decay of I Ba was fitted with a two-exponential function as described under "Experimental Procedures," and slow , fast and A s /A f were derived. The r400 values were calculated in the same way as in Fig. 5 the ␤ 2c subunit.
We could not conclude that the ␤ 2a subunit was not expressed in heart. In the present study, Northern blot analysis detected the 6-and 2-kb transcripts in heart with the ␤ 2aspecific probe. The identities of these transcripts were not clear. Theoretically, they might have been fragments of the ␤ 2a subunit. If so, our PCR analysis should have detected some ␤ 2a signals not only from brain but also from heart. If the 6-kb transcript was the mRNA of the ␤ 2 subunit that had not yet been processed, i.e. pre-mRNA, we could explain some of these results. However, we could not explain the 2-kb transcript. A part of the N terminus of the ␤ 2a subunit, which does not have the sequence of the forward primer, i.e. the total-beta-2-primer414f, might be expressed in heart, although 5Ј-RACE did not disclose any truncated forms of the ␤ 2a subunit. Otherwise, the 6-and 2-kb transcripts may be artifacts.
It is certain that the mechanism responsible for the slow inactivation occurring when the ␤ 2a subunit is expressed along with ␣ 1c subunit in heterologous expression systems, if indeed the former is a cardiac subunit, has not yet be clarified. When the ␤ 2c subunit was expressed along with the ␣ 1c subunit and the ␣ 2 ␦ subunit, the inactivation rates of I Ba were comparable with those from cardiac myocytes, although those with the ␤ 2a subunit were significantly slower, as reported previously (15). These observations suggest that the ␤ 2c subunit may be one of the functional ␤ 2 subunits in rat heart. However, we cannot deny that other ␤ subunits and/or other unknown regulators may also function in cardiac myocytes. Indeed, we could not reconstitute the native Ca 2ϩ channel, although we used all four subunits, i.e. ␣ 1c , ␤ 2c , and ␣ 2 ␦ subunits, which were cloned from rat heart. The sustained components of I Ba recorded from native cardiac myocytes (the r400 values) were significantly greater than those from BHK cells with ␤ 2c subunit. Moreover, the I-V relationships between the recombinant Ca 2ϩ channels and the native cardiac ones are significantly different. Furthermore, the inactivation kinetics of I Ca derived from recombinant Ca 2ϩ channels containing ␤ 2c and native channels were significantly different, although those of I Ba were similar. These data strongly indicate the existence of other missing auxiliary subunits or modulators, some of which are Ca 2ϩ -dependent. Alternatively, activity of modulators of Ca channel, such as calmodulin (28), in native cardiac myocytes might be different from that in BHK cells.
Cens et al. (29), using a series of deletion mutants and chimeric constructs of ␤ 1 and ␤ 2 subunits, showed that the N-terminal region of the ␤ 2a subunit was the major element slowing the inactivation. They found that a residual current at the end of a test pulse was large only if the N-terminal region of the chimeric constructs was derived from ␤ 2a . Our findings also confirmed that the short N-terminal region was important for modification of inactivation kinetics, because ␤ 2a and ␤ 2c subunits were only different in this region. The N-terminal region of the rat ␤ 2c subunit was identical to those of a Lambert-Eaton myasthenic syndrome antigen and the human ␤ 2c subunit, except for some amino acids considered to be species differences (23,30). The similarity to the former protein raises the possibility that the rat ␤ 2c subunit was cloned from neuron, which innervated into heart. However, this antigen was cloned from a fetal brain library using the serum from patients with Lambert-Eaton syndrome. Thus, it is possible that the antibody reacts with a region other than the N terminus. Moreover, Verschuuren et al. (31) reported that immunization of rats with the purified antigen induced high antibody titers, but no sign of neurological dysfunction was found. This observation implies that the antibody was not a cause of muscle weakness and did not cross-react with ␤ 2 subunits that resided in a neuromuscular junction. Therefore, it cannot be claimed that the antigen, i.e. ␤ 2c subunit, exists in the neuronal termini. On the contrary, its similarity to the latter, i.e. the human ␤ 2c subunit, indicates that the expression of the ␤ 2c subunit in heart is not specific to rat.
In human heart, mRNA expression of the ␤ 1b (3, 10), ␤ 1c (3, 10), and ␤ 3 (11) subunits and protein expression of the ␤ 2 subunit (12) have been demonstrated. In addition, the ␤ 2a (32) and ␤ 2c (23) subunits have been cloned from human heart. Among them, the N-terminal region of the rat ␤ 2c subunit was identical to the human ␤ 2c subunit except for some differences considered to be species-specific. However, the I-V relationship and the inactivation kinetics of the human ␤ 2c subunit reported were quite different from those of the rat ␤ 2c subunit (23). These differences may be caused by variations in the experimental conditions because Allen et al. (23) used Xenopus oocyte as the heterologous expression system and recorded I Ba at 15, 20, and 25°C, using 40 mM Ba 2ϩ as charge carrier. To our knowledge, there are no reports of studies directly comparing the inactivation kinetics of I Ba or I Ca from the recombinant cardiac L-type Ca 2ϩ channels composed of all four heteromultimeric subunits cloned from the heart of the same species, with those of native channels at a physiological temperature, i.e. 37°C.
In summary, the result of Northern blot analysis and the similarity of inactivation kinetics of the recombinant Ca 2ϩ channel using the ␤ 2c subunit with those of native cardiac myocytes strongly suggest that ␤ 2c subunit is one of the functional ␤ 2 subunits expressed in heart. Our findings also support the belief that the short N-terminal region of the ␤ 2 subunit is important for modification of Ca 2ϩ channel function and that different splice variants of the ␤ 2 subunit could modulate Ca 2ϩ entry through L-type Ca 2ϩ channels in different tissues and regions.