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Physiol. Genomics 17: 183-200, 2004. First published February 3, 2004; doi:10.1152/physiolgenomics.00207.2003
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Received 11 December 2003; accepted in final form 27 January 2004.
Physiological Genomics 17:183-200 (2004)
1094-8341/04 $5.00 © 2004 American Physiological Society

Molecular heterogeneity of calcium channel ß-subunits in canine and human heart: evidence for differential subcellular localization

Jason D. Foell 1, Ravi C. Balijepalli 1, Brian P. Delisle 1, Anne Marie R. Yunker 3, Seth L. Robia 2, Jeffrey W. Walker 2, Maureen W. McEnery 3, Craig T. January 1,2 and Timothy J. Kamp 1,2

1 Department of Medicine, University of Wisconsin, Madison 53792
2 Department of Physiology, University of Wisconsin, Madison, Wisconsin 53706
3 Division of General Medical Sciences, Case Western Reserve University, Cleveland, Ohio 44106


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Multiple Ca2+ channel ß-subunit (Cavß) isoforms are known to differentially regulate the functional properties and membrane trafficking of high-voltage-activated Ca2+ channels, but the precise isoform expression pattern of Cavß subunits in ventricular muscle has not been fully characterized. Using sequence data from the Human Genome Project to define the intron/exon structure of the four known Cavß genes, we designed a systematic RT-PCR strategy to screen human and canine left ventricular myocardial samples for all known Cavß isoforms. A total of 18 different Cavß isoforms were detected in both canine and human ventricles including splice variants from all four Cavß genes. Six of these isoforms have not previously been described. Western blots of ventricular membrane fractions and immunocytochemistry demonstrated that all four Cavß subunit genes are expressed at the protein level, and the Cavß subunits show differential subcellular localization with Cavß1b, Cavß2, and Cavß3 predominantly localized to the T-tubule sarcolemma, whereas Cavß1a and Cavß4 are more prevalent in the surface sarcolemma. Coexpression of the novel Cavß2c subunits (Cavß2cN1, Cavß2cN2, Cavß2cN4) with the pore-forming {alpha}1C (Cav1.2) and Cav{alpha}2{delta} subunits in HEK 293 cells resulted in a marked increase in ionic current and Cavß2c isoform-specific modulation of voltage-dependent activation. These results demonstrate a previously unappreciated heterogeneity of Cavß subunit isoforms in ventricular myocytes and suggest the presence of different subcellular populations of Ca2+ channels with distinct functional properties.

L-type calcium channel; splice variants


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
IN THE HEART, L-type Ca2+ channels play an essential role in multiple cellular processes including cellular excitability and excitation-contraction coupling. Voltage-gated L-type Ca2+ channels are multimeric complexes consisting of a pore-forming {alpha}1-subunit and auxiliary subunits including ß-, {alpha}2-{delta}-, and {gamma}-subunits (9). Previous studies have demonstrated that the {alpha}1C-subunit (Cav1.2) is the major {alpha}1-subunit present in adult ventricular muscle (9), and multiple splice variants have been identified (38). Another, even greater source for diversity for L-type Ca2+ channels in the heart is the expression pattern of the auxiliary subunits which finely modulate the properties of the expressed channels.

The Cavß subunit is a cytoplasmic protein that can be encoded by four different genes with multiple splice variants possible for each gene (6). The encoded proteins consist of five domains, and the two large central domains (D2 and D4) show high similarity between the gene products. However, the amino terminus (D1), small central linker (D3), and carboxy terminus (D5), exhibit much greater variability and are the sites for alternative splicing. The ultimate functional properties of the channel complex can be finely tuned depending on the Cavß isoforms present. Cavß subunit coexpression can shift the voltage dependence of channel activation and inactivation substantially (16, 55). Cavß subunits also play an essential role in voltage-dependent facilitation of currents through L-type Ca2+ channels (10, 35). One of the most prominent and potentially important roles of Cavß subunits is to act as a chaperone for trafficking Cav{alpha} subunits to the surface membrane, in part, by binding an endoplasmic reticulum retention signal (5, 12). In addition, there is emerging evidence that different ß subunits may allow targeting to different subcellular domains (7, 13, 15, 40, 60). Therefore, the ultimate functional properties and subcellular localization of L-type Ca2+ channels are dependent on the particular auxiliary ß-subunit isoforms present.

The Cavß subunit isoform expression pattern in the heart has been evaluated in previous studies, but no clear consensus has emerged in the literature. The rat Cavß2a isoform (GenBank accession no. M80545) was the first putative Ca2+ channel ß-subunit identified in the heart (47), and it was generally believed that cardiac L-type calcium channels included primarily the Cavß2a subunit (34, 47). However, the situation rapidly became more complex with the identification of multiple isoforms of the Cavß1 gene in samples from human heart (14). More recently, the Cavß3 gene has been found to be expressed in human heart (33). Differences between species may contribute to the confusion, but as additional studies have been performed even within a species consensus has not always emerged (63). Prior studies have often focused on defining the splice variants of a single Cavß gene and have not evaluated for the full range of possible Cavß isoforms. Although differential splicing of the four different Cavß subunit genes has been observed in neuronal tissues (22, 41, 59), little work has focused on systematically identifying the expression profile for all Cavß genes and their splice variants expressed in the human or canine heart.

The purpose of the present study was, first, to define the isoforms of the Cavß subunits expressed in canine and human heart using an RT-PCR strategy made possible by sequence information from the Human Genome Project and published data. Secondly, we evaluated for differential subcellular localization of the different Cavß isoforms by using isoform-specific antibodies. Finally, we determined the functional effects of previously uncharacterized Cavß2c isoforms on heterologously expressed Cav1.2 channels. A preliminary report of these findings has been made (20).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

Genomic structure and similarity of Cavß subunits.
Intron/exon structure of the four Cavß subunit genes was identified by BLAST alignments of Cavß cDNAs (GenBank) with the Human Genome Project draft sequence (Table 1). Similarity of individual exons from each Cavß gene was determined by aligning the translations using PileUp [Genetics Computer Group (GCG), Accelrys, San Diego, CA], and the percent similarity was determined using the OldDistances program in GCG.


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  		Table 1. Genomic structure of voltage-gated calcium channel Cavß subunits

 
RNA preparation and cDNA synthesis.
Canine hearts were obtained using a protocol conforming to the Guide for the Care and Use of Laboratory Animals published by the National Research Council (1996). Cardiac myocytes were isolated enzymatically from adult canine left ventricle as previously described (28). mRNA was prepared from isolated canine cardiomyocytes using the FastTrack 2.0 system (Invitrogen, Carlsbad, CA). Human tissue was obtained from donor hearts rejected for transplant due to technical reasons, following a protocol approved by the University of Wisconsin Human Subjects Committee. Human total RNA was isolated from 1 g of left ventricular tissue using RNAzol B solution (Tel-Test, Friendswood, TX). Reverse transcription was performed on both canine mRNA and human total RNA using random hexamers and the SuperScript First-Strand Synthesis System (Life Technologies, Rockville, MD).

Polymerase chain reaction.
PCR primers were designed to identify all known splice variants of the Cavß genes based on the published cDNAs and genomic structure from the Human Genome Project draft sequence. Oligonucleotide primers were synthesized by Life Technologies. Primer sequences are shown in Table 2, and primer pairs, amplicon sizes, and predicted protein lengths are shown in Table 3. All PCR experiments in canines were performed with cDNA synthesized from isolated left ventricular myocytes. Polymerase chain reactions contained 5.0 µl of cDNA from the reverse transcription reaction as template, 20 mM Tris·HCl (pH 8.8), 10 mM KCl, 10 mM (NH4)2SO4, 2.0 mM MgSO4, 0.1% Triton X-100, 0.1 mg/ml BSA; 2.0 µM each of dATP, dCTP, dGTP, and dTTP; 75 pmol of each primer, 5 U Taq Extender additive (Stratagene, La Jolla, CA), and 25 U Taq DNA polymerase (Fisher Scientific, Fair Lawn, NJ). PCR reactions were thermalcycled starting with an initial denaturation at 94°C for 3 min, cycled at 94°C for 45 s, 55°C for 45 s, and 72°C for 2 min for 45 cycles, and followed by 72°C for 7 min.


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  		Table 2. RT-PCR primer sequences

 

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  		Table 3. Isoform-specific RT-PCR primer pairs, amplicon sizes, amino acids amplified, and predicted protein length

 
Cloning and sequencing.
Amplified cDNA fragments were analyzed by 1.5% agarose gel electrophoresis and visualized by ethidium bromide staining under UV light. Each fragment was then purified from the agarose gel using the QIAquick Gel Extraction Kit (Qiagen, Valencia, CA). cDNA fragments were cloned into pCR 2.1-TOPO using the TOPO TA Cloning method (Invitrogen). Cloned RT-PCR fragments were cycle sequenced using BigDye chemistry (Perkin-Elmer, Foster City, CA) and automated analysis (University of Wisconsin Biotechnology Center, Madison WI). Briefly, cycle sequencing was performed using 5.0 pmol T7 primer, 10 µl of cloned RT-PCR fragment in pCR 2.1-TOPO vector, 4.0 µl BigDye buffer, and 4.0 µl BigDye. Sequencing reactions were denatured at 95°C for 3 min and then cycled at 95°C for 20 s, 45°C for 30 s, and 60°C for 2 min for 35 cycles, and followed by 72°C for 7 min. DNA sequence identities were verified by aligning with previously cloned Cavß subunits in GenBank and genomic sequences in the Human Genome Project using BLAST (1). Complete reading frames of Cavß2cN1, Cavß2cN2, Cavß2cN4, and Cavß2aN4 were amplified from human cDNA and cloned into pcDNA3.1HisTOPO (Invitrogen) using the following primers: fB2mik1v2 + rB2c and fB2c + rB2cterm.1 (Cavß2cN1, GenBank accession no. AY393860), fB2N5v2 + rB2c and fB2c + rB2cterm.1 (Cavß2cN2, AY393861), fB2UTR1.1 + rB2c and fB2c + rB2cterm.1 (Cavß2cN4, AY393862), and fB2UTR1.1 + rB2aex7A and fB2aex7A + rB2cterm.1 (Cavß2aN4, AY393859).

Cell culture and transfection of HEK 293 cells.
Low-passage HEK 293 cells were maintained in DMEM supplemented with 10% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin, and 2 mM L-glutamine. HEK 293 cells were transfected with either Cav1.2 full-length rabbit cardiac subunit (43), except for alternative splicing in domain IV S3 (56) cloned into pGW1H (British Biotechnology, Oxford, UK), rabbit skeletal muscle Cav{alpha}2{delta}-1 (17) cloned into pGW1H, pSV40TAg to increase expression levels, and GFPpRK5 expressing the S65T bright green fluorescent protein mutant only or with Cavß2cN1, Cavß2cN2, Cavß2cN4, or Cavß2aN4 using the calcium phosphate transfection method (Invitrogen). Briefly, 10 µg of total cDNA were transfected into HEK 293 cells and incubated for 4 h. Cells were washed four times with PBS and incubated overnight in DMEM.

Electrophysiology.
Whole cell recordings were performed within 24 h after transfection. External solution consisted of (in mM) 10 BaCl2, 133 CsCl, and 10 HEPES (pH 7.4 with 1 N CsOH). Internal solution consisted of (in mM) 114 CsCl, 10 EGTA, 10 HEPES, and 10 Mg-ATP (pH 7.2 with 50 mM CsOH). Borosilicate glass pipettes were pulled to a resistance of 1–2.5 M{Omega} when filled with internal solution. Membrane capacitance and series resistance were compensated to at least 70%. Whole cell currents were recorded using an Axopatch 200B amplifier sampled every 40 ms and filtered through a low-pass filter at 5 kHz (Axon Instruments, Foster City, CA). Current-voltage (I-V) protocols consisted of a holding potential of –80 mV pulsing in steps of 10 mV to +70 mV for 50–400 ms and repolarizing to –80 mV. Leak and capacitive currents were subtracted using a P/4 protocol.

Whole cell conductance (G) was calculated from the peak inward IBa divided by the difference of the test potential and the estimated reversal potential of +60 mV. The G-V data were fit to a Boltzmann distribution according to the following equation

where Gmax is the maximal whole cell conductance, V1/2 is the voltage midpoint for the distributions, and k is the slope factor. The data were fit using nonlinear least squares regression analysis with the Levenberg-Marquardt or Simplex methods available with Origin 6.0 (Microcal, Northampton, MA).

Fluorescence confocal microscopy.
Immunolabeling was performed on isolated canine left ventricular myocytes using the following primary antibodies: rabbit polyclonal antibodies to Cav1.2 (29), Cavß1b antibody CW28 (58), Cavß2 antibody CW48 (58), Cavß3 (Alomone Labs, Jerusalem, Israel), and Cavß4 antibody CW34 (58), and a guinea pig polyclonal antibody to Cavß1a (61). Isolated myocytes were initially fixed with 2% buffered paraformaldehyde for 10 min. Fixed cells were permeabilized with Triton X-100 (0.1%) for 10 min and then quenched for aldehyde groups in 0.75% glycine buffer for 10 min. After washing with TBS (two 10-min washes), cells were incubated with 1 ml blocking solution (2% BSA and 2% goat serum, 0.05% NaN3 in TBS) for 2 h with gentle agitation at 4°C to block nonspecific binding. Subsequently, cells were incubated overnight with respective primary antibodies in blocking solution at 4°C. Antibody dilution of primary antibodies was 1:100 for the polyclonal anti-Cav1.2, anti-Cavß1a, anti-Cavß2, anti-Cavß3, and anti-Cavß4, and a 1:500 dilution of anti-Cavß1b. Excess primary antibody was washed off with the use of blocking solution (three 1-h washes). The cells were then incubated overnight with Alexa-conjugated secondary antibodies (Molecular Probes, Eugene, OR; 2 mg/ml) diluted 1:200 in blocking solution. Highly cross-absorbed Alexa 568 goat anti-rabbit IgG (H+L) and Alexa 488 goat anti-guinea pig (H+L) were used. The cells were then washed with blocking solution (three 2-h washes), resuspended in blocking solution, and mounted on a coverslip. To determine nonspecific binding, control experiments with secondary antibody alone were also performed.

Imaging was performed with a Bio-Rad MRC 1024 laser-scanning confocal microscope equipped with a mixed gas (Ar/Kr) laser operated by 24-bit LaserSharp software (Bio-Rad, Hercules, CA). Image acquisition in the green channel utilized excitation at 488 nm with emission detected at 522 ± 17 nm. Acquisition in the red channel utilized excitation at 568 nm with emission detected at 605 ± 16 nm.

Membrane fractionation.
Sarcolemmal, T-tubular, and dyadic membrane fractions were prepared by the methods described previously (2). Briefly, portions of canine left ventricular tissue were homogenized and subjected to a series of differential centrifugations. Isolated canine left ventricular myocytes and human left ventricular tissue were also fractionated and analyzed on Western blots (data not shown). High-salt-washed membranes were layered on a discontinuous sucrose density gradient of 21, 31, 40, and 55% sucrose and centrifuged for 2 h at 141,000 gmax. Discontinuous density gradient centrifugation produced three distinct interfaces at 10/21%, fraction 1 (F1) enriched in surface sarcolemmal membrane; 21/31%, fraction 2 (F2) enriched in T-tubule membrane; and 31/40%, fraction 3 (F3) enriched in junctional complexes. Interfaces were pelleted at 141,000 gmax and suspended with the protease inhibitors 0.1 µg/ml leupeptin, 0.1 µg/ml pepstatin, and 1 µg/ml aprotinin and stored at –80°C. Protein concentrations were determined by the Lowry method.

SDS-PAGE and relative quantitative Western analysis.
Membrane proteins (60 µg) from each of the membrane fractions from both human and canine hearts were separated by SDS-PAGE using 7.5% bis-acrylamide gels as described by Laemmli (39). Membrane protein (20–60 µg of protein) was solubilized in sample buffer (62.5 mM Tris·HCl, pH 6.8, 10% glycerol, 2% SDS, 5% 2-mercaptoethanol, and 0.1% bromophenol blue) by warming to 60°C for 30 min prior to loading onto the gel. Following separation, proteins were transferred to nitrocellulose membrane (Schleicher and Schuell, Dassel, Germany) by blotting for 1 h at 105 V. Nonspecific binding sites were blocked by immersion of membranes overnight at 4°C in PBS detergent (0.1%, Tween-20) containing 5% (wt/vol) dried skim milk. Membranes were then probed with primary antibodies with the following dilutions: 1:500 for polyclonal anti-Cav1.2 and anti-Cavß1b; 1:200 for anti-Cavß2, anti-Cavß3, and anti-Cavß4; and 1:1,000 for anti-Cavß1a. Donkey anti-rabbit immunoglobulin linked to horseradish peroxidase (1:50,000) detected bound antibody for Cav1.2, Cavß1b, Cavß2, Cavß3, and Cavß4. Goat anti-guinea pig immunoglobulin conjugated to peroxidase (Sigma, St. Louis, MO) was diluted 1:30,000 to detect Cavß1a. Immunoreactivity was visualized using peroxidase-based chemiluminescent detection system, ECL (Amersham Life Sciences, Cleveland, OH). Relative quantitation of Western blots was accomplished using a Bio-Rad model GS-700 image densitometer. Conditions were optimized for each antibody by testing a range of antigen loading (20–120 µg of membrane protein) and determining the resulting densitometric signal. The relationship between antigen loaded and resulting densitometric signal was found to be linear over most of the tested concentration range. For all antibodies tested, at 60 µg of protein loaded, the signal was in the linear range. Multiple exposure times of the autoradiograms were also performed to optimize linearity and avoid signal saturation.

Statistics.
All values are presented as mean ± SE. Statistical significance was evaluated by the Student’s unpaired t-test. For multiple comparisons, analysis of variance (ANOVA) was performed. Differences with P < 0.05 were considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 

Identification of Cavß1 splice variants.
The results of the BLAST alignment of the Cavß1 cDNAs to the human genomic DNA show that the Cavß1 gene is composed of 15 exons of which only 14 are transcribed with exons 7A and 7B being alternatively spliced (see Table 1 and Figs. 1 and 4). This analysis is in agreement with the genomic structure of the Cavß1 gene determined in a previous study using human genomic clones, with the exception of exons 13 and 14, which were described as alternative exons 13a and 13b previously by Hogan et al. (32). This minor discrepancy lies in the fact that our comparison of cDNA with the Human Genome Project sequence demonstrates that exons 13 and 14 are not alternative exons, and in the case of Cavß1b, an alternative splice donor site in exon 13 is used to splice with exon 14.



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  		Fig. 1. RT-PCR of Cavß1 splice variants in human and canine left ventricle. A: at top is a schematic of the 15 exons of the Cavß1 gene, and below this are diagrams depicting the four splice variants amplified from canine and human left ventricle. The gray scale of each exon box denotes the similarity across the four Cavß genes. Regions of greatest variability (6–34% similarity) are white, boxes shaded in light gray are exons of intermediate similarity (53–82%), and the boxes in dark gray mark exons with a high degree of similarity (71–100%). RT-PCR primers used to specifically amplify each splice variant are denoted with arrows. Alternatively spliced exons are denoted by peaked lines ("^"), and truncations are denoted by an asterisk (in Cavß1d). Antibody epitopes are indicated by "Ab" above a bold line. B: RT-PCR products identifying each of the four splice variants with the actual size of amplified products of the Cavß1 gene in human and canine left ventricle.

 


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  		Fig. 4. Human Cavß1 subunit amino acid sequence alignment (shaded regions identify regions of high similarity). Alignment show the Cavß1 splice variants expressed in the heart including Cavß1a (accession no. M92301), Cavß1b (M92303), Cavß1c (M92302), and Cavß1d (AY393857).

 
Initial screening for Cavß1 transcripts was performed using primers flanking the alternatively spliced exon 7 using a forward primer in exon 3 (fB1) and a reverse primer in exon 8 (rB1) with mRNA prepared from isolated canine ventricular myocytes. Multiple fragments were amplified, isolated, and sequenced. These RT-PCR products were identified as Cavß1a (exon 7A), Cavß1b/Cavß1c (exon 7B), and a novel splice variant that did not contain exon 7A or 7B, denoted Cavß1d. The identification of Cavß1d was difficult because the difference in the Cavß1b/Cavß1c and Cavß1d products is only 20 bp, which is beyond the resolution of typical agarose gel electrophoresis, and thus the existence of Cavß1d was only appreciated after screening and sequencing of multiple clones. Isoform-specific primers were then used to amplify Cavß1a, Cavß1b, and Cavß1c, which were previously identified splice variants of the Cavß1 gene (14, 48, 53), as well as the novel splice variant Cavß1d (Fig. 1). The identity of all four Cavß1 splice variant RT-PCR products was verified by sequencing. The Cavß1a contains exon 7A, whereas Cavß1b and Cavß1c have exon 7B. In contrast, Cavß1d skips exon 7A and 7B, which shifts the reading frame, resulting in a premature stop site in exon 8. As seen in Fig. 1A, both Cavß1a and Cavß1c have the same carboxy terminus ending after exon 13, but Cavß1b uses an alternate splice site in exon 13 to which exon 14 is spliced to form the carboxy terminus. No splice variations were identified at the amino terminus of the Cavß1 gene using specific NH2-terminal primers for RT-PCR. Additional experiments using total RNA isolated from human left ventricle also revealed the same four splice variants of the Cavß1 gene with differential splicing restricted to exon 7 and the carboxy terminus at exons 13 and 14 (Fig. 1 and Table 1). Previous studies of human heart have also detected Cavß1a, Cavß1b, and Cavß1c, but not Cavß1d (14). Figure 4 shows the deduced amino acid sequences for the four identified Cavß1 isoforms.

Identification of Cavß2 splice variants.
The Cavß2 gene has been suggested to encode the predominant Cavß isoform(s) expressed in the heart; however, there is little information on the gene structure and extent of splice variants expressed. The genomic structure of the Cavß2 gene was first determined by aligning the draft sequence from the Human Genome Project to the known Cavß2 cDNAs in GenBank demonstrating 20 different exons (Table 1). The complex splicing of the Cavß2 gene is evident by the five different amino terminal splice variants. We refer to these five different NH2 termini as N1 (exon 1A + exon 2A), N2 (exon 1B + exon 2A), N3 (exon 2B), N4 (exon 2C), and N5 (exon 2D). The middle of the gene is also spliced with four alternative splices with either exon 7A, exon 7B, a unique exon 7C, or no exon 7. In keeping with the current nomenclature of the ß-subunits, these are termed as Cavß2a (exon 7A), Cavß2b (exon 7B), Cavß2c (exon 7C), and Cavß2d (no exon 7). Then to expand the nomenclature to include the NH2-terminal splice variants, we use the designations Cavß2aN1, Cavß2aN2, Cavß2aN3,...etc. Thus the differential combination of the five possible NH2-terminal exons with the four different exon 7 splices yields at least 20 possible Cavß2 splice variants.

Specific primers for RT-PCR were used to amplify splice variants of the Cavß2 gene from canine and human heart as shown in Fig. 2. We have identified 9 of the possible 20 splice variants of the Cavß2 gene present in both the human and canine heart: Cavß2aN1, Cavß2aN2, Cavß2aN4, Cavß2aN5, Cavß2bN4, Cavß2cN1, Cavß2cN2, Cavß2cN4, and Cavß2dN4. Additionally, the Cavß2bN4, Cavß2cN1, Cavß2cN2, Cavß2cN4, and Cavß2dN4 represent five novel isoforms not previously identified in any tissue. The Cavß2aN4 has previously been identified in the rabbit and human heart (34, 57), Cavß2aN2 in the rabbit heart (34), Cavß2aN1 in the rat heart (63), and Cavß2aN5 in mouse heart (42). The palmitoylated amino-terminal splice variants (Cavß2N3) were not identified in either the human or canine heart. Also, no differential splicing was identified at the COOH terminus (from exons 8–14) of the Cavß2 gene. Figure 5 shows the amino acid sequence alignments for the Cavß2 splice variants identified in the human heart.



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  		Fig. 2. RT-PCR of Cavß2 splice variants in human and canine left ventricle. A: at top is a schematic depiction of the 20 exons of the Cavß2 gene, and below this the 9 splice variants detected in human and canine left ventricle are shown schematically with arrows underneath denoting primer pairs. Exons 10–12 are represented by "...". The shadings of the exon boxes and other symbols are as described for Fig. 1. B: RT-PCR products amplified from the 9 splice variants with the actual size of amplified products transcribed from the Cavß2 gene in human and canine left ventricle.

 



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  		Fig. 5. Human Cavß2 subunit amino acid sequence alignment (shaded regions identify regions of high similarity). Alignment shows Cavß2 splice variants expressed in the heart with Cavß2aN1 (AF423191), Cavß2aN2 (AY393858), Cavß2aN4 (AY393859), Cavß2aN5 (AF423192), Cavß2bN4, Cavß2cN1 (AY393860), Cavß2cN2 (AY393861), Cavß2cN4 (AY393862), and Cavß2dN4 (AY393863). *Cavß2bN4 has been cloned only through exon 7B.

 
Identification of Cavß3 and Cavß4 splice variants.
We next determined whether isoforms of the Cavß3 and Cavß4 genes are expressed in the canine and human left ventricle. Previous studies have identified the intron/exon structure and the splice variants of both the human and murine Cavß3 gene (44, 62). The results of the human genomic BLAST alignments to the human Cavß3 cDNA show a similar intron/exon structure and with the same sized exons as previously shown in the human. Table 1 shows the 13 coding exons for the Cavß3 gene from the Human Genome Project draft sequence (accession no. NT_029419.10). The numbering of the exons for the Cavß3 gene is different from the other three Cavß subunit genes because the Cavß3 exon 2 is homologous with the exon 3 from Cavß1, Cavß2, and Cavß4 genes. Thus the corresponding numbers for the homologous exons are shifted down so that exon 6 of the Cavß3 gene is homologous to exon 7B in Cavß1 and Cavß2 and to exon 7 for Cavß4 (Table 1). BLAST searches of this genomic clone do not identify homologous exons to either exon 7A in Cavß1 and Cavß2 or exon 7C in Cavß2.

Utilizing isoform-specific primers, a single splice variant (Cavß3b), containing all 13 exons of the Cavß3 gene, was amplified from canine and human ventricle (Fig. 3A). This observation is consistent with other studies showing that the Cavß3b isoform is expressed in human and rabbit heart and human brain (14, 33, 34, 44). Unlike the Cavß1, Cavß2, and Cavß4 genes, the "d" variant, Cavß3d, of the Cavß3 gene was not detected in either canine or human heart. However, Cavß3d has been previously identified in murine stem cells and more recently in the human heart (33, 44). RT-PCR screening of the amino and carboxy termini of the Cavß3 gene did not detect additional splice variants.



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  		Fig. 3. RT-PCR of Cavß3 and Cavß4 splice variants in human and canine left ventricle. A: schematic display of the 13 exons of the Cavß3 gene with the detected single splice variant (arrows underneath denote primer pair), with Cavß3b depicted along with the actual size of the RT-PCR product from canine and human left ventricle. B: comparison showing the 15 exons of the Cavß4 gene, with the four lower diagrams corresponding to the four splice variants detected (arrows underneath denoting primer pairs). The shadings of the exon boxes and other symbols are as described for Fig. 1. The bottom of B shows actual sizes of the four splice variants amplified by RT-PCR.

 
The intron/exon structure of the human Cavß4 gene has not previously been described. Table 1 shows the 15 coding exons for the Cavß4 gene from the Human Genome Project draft sequence (accession no. NT_005403.13). BLAST searches of the Cavß4 genomic clone did not identify homologous sequences to either exon 7A from Cavß1 and Cavß2 genes or exon 7C in Cavß2. Isoform-specific primers were generated based on the human genomic alignments to the cloned Cavß4 cDNAs. Initially, nonspecific Cavß4 primers in exon 3 and specific primers to exon 7 and the junction of exons 6 and 8 were used to amplify Cavß4b and Cavß4d splice variants. Recent studies by Helton et al. (30) have shown that there are at least two NH2-terminal splices in the human brain including a novel short amino terminus (Cavß4N2) that is homologous with the NH2 terminus of Cavß3. Utilizing this information, we used specific primers to each Cavß4 NH2 termini in conjunction with Cavß4b- and Cavß4d-specific primers to amplify four different isoforms of the Cavß4 gene. Figure 3B shows the four splice variants of the Cavß4 gene detected in canine and human ventricle: Cavß4bN1 and Cavß4bN2, which include exon 7; and Cavß4dN1 and Cavß4dN2, which are missing exon 7. Cavß4bN1 was first cloned from the rat brain and subsequently in the human brain (GenBank accession no. U95020) (8). Subsequent studies have identified Cavß4b in human cerebellum and temporal lobe using riboprobes and antibodies that are directed at the COOH terminus of Cavß4b and thus do not differentiate between Cavß4bN1 and Cavß4bN2 (40, 59). However, no studies to date have identified Cavß4bN1 in the human or canine heart. Studies by Hibino et al. (31) have identified by RT-PCR a truncated Cavß4 splice variant in the chicken heart. Additionally, the Cavß4 gene showed no differential splicing at the COOH terminus. Figure 6 shows the aligned amino acid sequences for the Cavß3 and Cavß4 isoforms.



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  		Fig. 6. Human Cavß3 and Cavß4 subunit amino acid sequence alignments (shaded regions identify regions of high similarity). Alignments show Cavß3b (X76555) and Cavß4bN1 (U95020), Cavß4dN1, Cavß4bN2 (AY054985), and Cavß4dN2 splice variants expressed in the heart. *Human Cavß4dN1 was cloned from NH2-terminal sequences specific for Cavß4N1 starting in exon 2A through the termination codon.

 
Identification and subcellular localization of Cav1.2 and Cavß proteins.
To verify that the Cavß isoforms identified by RT-PCR were expressed as proteins, Western blot analysis and immunocytochemistry were performed with isoform-specific antibodies directed at Cav1.2, Cavß1a, Cavß1b, Cavß2, Cavß3, and Cavß4. Furthermore, the subcellular distribution of these proteins was evaluated by semiquantitative Western blot analysis of different membrane fractions as well as by confocal microscopy of immunolabeled myocytes. Western blot analysis was performed on a membrane homogenate as well as three sucrose density gradient fractions enriched in surface sarcolemma (F1), T-tubular sarcolemma (F2), and junctional complexes (F3) (2). As shown on the Western blots in Fig. 7, the sucrose density gradients greatly enrich the membranes containing the Cav1.2 and Cavß subunits relative to crude homogenate and provide more ready detection of these relatively low-abundance proteins.



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  		Fig. 7. Western blot and densitometric analysis of Cav1.2 and Cavß subunits, displaying the results for a given subunit-specific antibody with a representative Western blot of discontinuous gradient fractions enriched in surface sarcolemma (F1), T-tubular sarcolemma (F2), and junctional complexes (F3), as well as membrane homogenate (H). The normalized, average data for densitometric scanning of each series of immunoblots are provided in the bar diagram (n = 3–7, mean ± SE; Hgt = homogenate). A–F: antibodies directed at Cav1.2, Cavß1a, Cavß1b, Cavß2, Cavß3, and Cavß4, respectively.

 
To determine the subcellular localization of the Cav{alpha} subunit in canine ventricle, immunoblots utilizing an antibody directed against Cav1.2 were performed. A doublet of 190 and 240 kDa was detected with the greatest abundance present in F2 (Fig. 7A), suggesting a strong T-tubular presence of this protein. This concurs with immunocytochemistry showing an ordered, punctate staining pattern typical of T-tubular staining (Fig. 8A). These results are also consistent with dihydropyridine binding (3H-PN200–110) of these fractions reported previously, with the greatest binding in the T-tubular fraction (2).



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  		Fig. 8. Subcellular localization of Cav1.2, Cavß1a, Cavß1b, Cavß2, and Cavß4 in canine left ventricular myocytes. Confocal images of isolated myocytes immunolabeled with primary antibodies directed against Cav1.2 (A), Cavß1a (B), Cavß1b (C), Cavß2 (D), and Cavß4 (E).

 
Western blot analysis and immunocytochemistry were then performed with Cavß isoform-specific antibodies. First, we tested for the presence of Cavß1a and Cavß1b protein. Western blots probed with anti-Cavß1a antibody showed staining of major bands at 58 and 64 kDa in all three enriched membrane fractions with the greatest abundance in F1 (Fig. 7B). This is similar to a 52/62-kDa doublet and a 71-kDa minor band described previously from crude homogenates of rabbit skeletal muscle where Cavß1a is the predominant Cavß subunit (61). However, the Cavß1a was not detected at the protein level in rabbit heart using the same antibody (61). Immunocytochemistry with anti-Cavß1a antibody revealed a distinct staining pattern compared with Cav1.2 staining, with predominantly surface sarcolemmal staining (Fig. 8B). In contrast, Western blots probed with anti-Cavß1b antibody detected 66-kDa and 75-kDa proteins with the strongest signal in F2, the T-tubular enriched fraction (Fig. 7C). This pattern was quite similar to the proteins of 66 and 75 kDa specifically detected in HEK 293 cells transfected with Cavß1b (GenBank accession no. M92303) cloned from the human brain (data not shown). This compares to an 82-kDa band detected in rat brain with this antibody (58) and a 75-kDa protein detected in canine brain (data not shown). The predicted size from the cDNA sequence is 66 kDa, suggesting possible differences in posttranslational processing between the tissues. The corresponding immunocytochemistry of Cavß1b shows preferential staining of the T-tubules in canine cardiomyocytes (Fig. 8C).

Nine different isoforms of the Cavß2 gene were identified using RT-PCR with expected protein molecular masses of 64–74 kDa. However, there are not specific antibodies available for each of these isoforms, so we utilized a Cavß2 antibody that recognizes the common COOH terminus present on eight of the nine (Cavß2dN4 is truncated and does not express the epitope). Anti-Cavß2 antibody identified a predominant 75-kDa band on Western blots of all membrane fractions with the greatest signal from F2 (Fig. 7D). However, with longer exposure, Western blots revealed a range of sizes from 65–85 kDa. Previous studies have shown sizes of 62–100 kDa in canine, rabbit, porcine, and human heart and thus support the identification of multiple isoforms of the Cavß2 gene (21, 25, 26, 51, 61). Longer exposures of blots also revealed a clear band in the crude homogenate lane not evident in the exposure on Fig. 7. Immunolabeled, isolated ventricular myocytes with anti-Cavß2 showed a distinct T-tubule staining pattern as well as some staining of surface sarcolemma (Fig. 8D).

RT-PCR experiments have shown that a single isoform of the Cavß3 gene was amplified in canine and human heart (Fig. 3A). The anti-Cavß3 antibody identifies a 58-kDa protein in all three of the enriched membrane fractions. This is similar to other reports showing that a 58-kDa protein is expressed in rabbit heart and a 63-kDa protein in porcine heart (51, 61). Membranes from F2 showed the greatest Cavß3 immunoreactivity, consistent with the highest abundance of the protein in T-tubule membranes (Fig. 7E). The anti-Cavß3 antibody failed to give specific immunolabeling of isolated myocytes.

The results of the RT-PCR show the presence of four isoforms of the Cavß4 gene, Cavß4bN1, Cavß4bN2, Cavß4dN1, and Cavß4dN2. The anti-Cavß4 antibody used specifically recognizes an epitope on the COOH terminus of Cavß4 and so would not be expected to detect Cavß4dN1 or Cavß4dN2. Western blots using anti-Cavß4 antibody revealed a single 45-kDa band in canine membrane fractions. The predicted molecular mass based on the amino acid sequence for each Cavß4 splice variant is 58 kDa (Cavß4bN1), 22 kDa (Cavß4dN1), 55 kDa (Cavß4bN2), and 19 kDa (Cavß4dN2). The Cavß4 Westerns showed the greatest predominance of this protein by far in the surface sarcolemma-enriched F1 (Fig. 7F). With longer exposures of blots, bands were also seen in F3 and homogenates (data not shown). In agreement with the membrane fractionation studies, immunolabeling of the isolated myocytes with the anti-Cavß4 antibody showed preferential staining of the surface sarcolemma (Fig. 8E). Previous studies have not detected Cavß4 immunoreactivity in the adult ventricular muscle from rabbit (21, 61).

Experiments were also performed to verify the presence of these Cavß proteins in human heart. Enriched membrane fractions were probed with the same panel of antibodies directed against Cavß1a, Cavß1b, Cavß2, Cavß3, and Cavß4. Immunoreactivity was found specifically for each antibody with the identified proteins of comparable molecular weight to those found in canine heart (data not shown). The scarcity of human tissue made extensive membrane fractionation studies not feasible.

As a test to confirm that the immunoreactivity observed on Western blots was due to proteins present in cardiomyocytes, each antibody was tested on enriched membrane fractions made from enzymatically isolated canine ventricular myocytes. These preparations had no identifiable cells types except ventricular myocytes and should have minimal contamination. The panel of antibodies, likewise, recognized proteins of identical size to those detected in left ventricular tissue membrane preparations, confirming protein expression of all four Cavß genes in ventricular myocytes (data not shown).

Electrophysiology of novel Cavß2c isoforms.
The newly identified Cavß2c isoforms are unique in containing the central exon 7C in contrast to exon 7A or 7B, and the functional properties of exon 7C containing Cavß2 subunits have not previously been determined. Exon 7C is of particular interest, as homologous exons have not been found in any of the other Cavß genes, unlike exons 7A and 7B, which share homology with the other Cavß genes. Therefore, we isolated full-length human heart clones for Cavß2cN1, Cavß2cN2, and Cavß2cN4 to compare with the well-characterized Cavß2aN4 isoform. Heterologous expression experiments were performed in HEK 293 cells coexpressing Cav1.2, Cav{alpha}2{delta}, and Cavß subunits using the whole cell patch-clamp technique. Representative raw current traces are shown for Cav1.2 + Cav{alpha}2{delta} and with each of the four cloned ß-subunits in Fig. 9A. The peak current density was increased in the range of 6- to 10-fold by coexpression of Cavß subunits with Cav1.2+Cav{alpha}2{delta} subunits as shown by the average I-V data in Fig. 9B. No significant differences between peak currents for the Cavß isoforms were detected with ANOVA analysis of the group. The voltage dependence of current activation was shifted in the hyperpolarizing direction by coexpression of Cavß subunits as shown by the negative shift of the peak of the I-V and more precisely shown by the activation curves calculated from the peak currents in Fig. 9C. Cavß2aN4, Cavß2cN2, and Cavß2cN4 all resulted in a comparable hyperpolarizing shift of the V1/2 for the Boltzmann fit activation curves compared with Cav1.2+Cav{alpha}2{delta} only (–14.1 ± 0.9, –12.3 ± 1.1, or –13.1 ± 1.0 vs. –5.4 ± 0.5 mV, respectively, with P < 0.05 for each comparison). In distinction, coexpression of Cavß2cN1 resulted in a significant shift in V1/2 (–8.3 ± 0.7 mV) compared with Cav1.2+Cav{alpha}2{delta} only, but this shift was significantly less than observed with the other Cavß subunits studied. Thus alternative splicing limited to only the amino terminus of Cavß2c can differentially impact voltage-dependent activation.



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  		Fig. 9. Functional effects of coexpressing Cavß2c subunits with Cav1.2 and Cav{alpha}2{delta}. A: representative raw current traces at –30 mV, +10 mV, and +30 mV recorded from transiently transfected HEK 293 cells with Cav1.2 and Cav{alpha}2{delta} alone and with either Cavß2cN1, Cavß2cN2, Cavß2cN4, or Cavß2aN4. B: average current-voltage relationships, with inset showing subunit combinations and number of experiments. C: normalized whole cell conductance (G) vs. voltage (V) relationships with data fit by Boltzmann distributions as shown by the solid lines. D: average ratio of current decay determined at a test potential of +10 mV with the current measured at 50, 200, and 400 ms relative to the peak current for the different subunit combinations. *P < 0.05.

 
Cavß subunits have also been previously demonstrated to have potent effects on channel inactivation. Therefore, we examined in detail current decay with and without Cavß subunit coexpression. Inspection of the representative current traces (Fig. 9A) or the average data (Fig. 9D) revealed that, as has been the case for most Cavß subunits, there is a significant acceleration of current inactivation. The ratio of the current remaining at 50, 200, and 400 ms relative to the peak current was used as a model independent measure of current decay. All four Cavß subunits studied resulted in a significant acceleration of current decay relative to Cav1.2+Cav{alpha}2{delta} alone (P < 0.05 for each, Fig. 9D). There was no statistically significant difference among the Cavß subunits themselves.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 

Using a systematic PCR screening strategy based on genomic sequence information made available by the Human Genome Project and published literature, we provide a detailed description of the rich diversity of Cavß subunits expressed in human and canine ventricular muscle, with 18 distinct isoforms identified. Six of these isoforms have not previously been identified in the heart, and three full-length splice variants of the Cavß2 gene (Cavß2cN1, Cavß2cN2, and Cavß2cN4) were cloned and functionally characterized for the first time. We also detected expression of all four known Cavß genes in the heart at the protein level using specific antibodies. Immunocytochemistry and membrane fractionation studies demonstrated distinctive patterns of subcellular distribution for different Cavß isoforms with some more localized to the T-tubules, and others showing increased presence in surface sarcolemmal regions. Based on these results, we will discuss why this diversity is only now being fully appreciated and the implications for ventricular myocyte cell function.

Uncovering diversity of Cavß isoforms.
The rich diversity of Cavß isoforms in the heart may have been overlooked in prior studies for a number of reasons. First, early studies were limited by the information available on splice variants and known genes. Additionally, certain techniques, such as Northern blots, cannot easily distinguish between many of the splice variants of a single gene. By identifying the intron/exon structure using genomic and isoform alignments with GCG and the Human Genome Project BLAST, we were able to design PCR primers that would allow us to detect all known splice variants for the four Cavß genes. At the protein level, the use of enriched membrane fractions improved the sensitivity of Western blots to detect relatively low-abundance Cavß proteins. The present study focused on canine and human ventricle that have highly similar Cavß isoform expression patterns, but other species, and particularly rodents such as mouse and rat, may have quite different Cavß expression profiles. Because we identified all 18 splice variants in isolated canine ventricular myocytes by RT-PCR, we believe this indicates that these splice variants are expressed specifically in myocytes. However, it is impossible to rule out a very low level of contaminating cell types. Results using intact human and canine ventricular myocardium which contain endothelial cells, fibroblasts, smooth muscle and neurons interestingly revealed an identical pattern of Cavß isoform expression suggesting either that no additional Cavß splice variants are present in these cell types or significant contamination of the isolated myocyte preparations had occurred. Ultimately, the diversity of Cavß subunits found in the heart may be even greater, as this study did not examine right ventricular or atrial tissue. Furthermore, distinct transmural patterns of Cavß subunit distribution may also be present.

The presence of the unique palmitoylated Cavß2aN3 in the heart has been controversial. It was first reported in the rat heart (47), but the probe used for Northern blot analysis was not specific for this Cavß isoform. Several subsequent attempts have been unsuccessful in identifying this isoform in the heart of a variety of species included in the present study for human and canine heart (50, 63). In addition, a detailed cellular electrophysiology study comparing heterologously expressed ß2a (Cavß2aN3) and ß2b (Cavß2aN4) subunits with native calcium currents in rat ventricular myocytes argued that the palmitoylated Cavß2aN3 was at least functionally absent in native ventricular myocytes, based on the kinetics of current decay (13). However, two recent studies have reported that the palmitoylated Cavß2aN3 is expressed in the human heart (33, 64). Based on the majority of molecular and functional data, we conclude that Cavß2aN3 is not expressed to a significant extent in human or canine ventricle.

Cavß subunit nomenclature.
Finding and describing 18 isoforms of the Cavß subunit required us to update the current ß-subunit nomenclature (18). Unfortunately, the literature is complicated by a variety of naming schemes for different Cavß subunit isoforms with many discrepancies. For example, the same group cloned "ß2a" from the rabbit and also later identified "ß2a" in the human heart, but these are different isoforms with distinct amino termini, Cavß2aN4 and Cavß2aN3, respectively (33, 34). As the list of splice variants has grown, particularly in the amino terminus of the Cavß2 and Cavß4, the existing nomenclature scheme is proving incomplete. We start by using the existing strategy of naming Cavß subunit splice variants based on alternatively spliced exons 7A, 7B, and 7C in the central variable region (D3) of Cavß2 protein by designating these isoforms by the gene number (2) followed by "a", "b", or "c", referring to the alternative exon or "d" if no exon (i.e., Cavß2a, Cavß2b, Cavß2c, Cavß2d). We add to this a designation of the amino terminus structure using an intuitive description of the numerous splice variants based on the order that they occur within the intron/exon structure of the gene. Therefore, the inclusion of the first exon (designated "1A" in Fig. 2) in the transcribed message is denoted "N1" (i.e., Cavß2aN1) and subsequent splicing patterns, as shown in Fig. 2, produce the other amino terminal variants (Cavß2aN2, Cavß2aN3, Cavß2aN4, and Cavß2aN5). This strategy has allowed us to uniquely identify each splice variant described in the present study and proves adequate to uniquely identify all of the currently known splice variants for Cavß genes.

Subcellular localization of Cavß subunits.
Cavß subunits are known to play an important role in the membrane trafficking of Ca2+ channel complexes based largely on data from heterologous expression systems. For example, Cavß subunits have been demonstrated to chaperone {alpha}1-subunits to the surface membrane when expressed in HEK 293 or Cos-7 cells (5, 12, 24, 36). There is also emerging evidence that different ß-subunits may allow differential targeting to subcellular domains in certain cell types, such as when Cavß subunits are heterologously expressed in a polarized epithelial tissue (7). In human hippocampus, a differential subcellular distribution of Cavß isoform immunoreactivity has been detected with Cavß1, Cavß2, and Cavß3 largely localized to neuronal cell bodies, whereas Cavß4 showed a more dendritic localization (15, 40). Recently, studies have suggested differential localization of ß-subunits in rat cardiomyocytes; however, these experiments detected exogenous expression of ß-subunits by transduction of rat heart cells with adenoviral constructs with ß1b, ß2a (Cavß2aN3), ß3, and ß4 fused with GFP (13, 60). The localization of exogenous ß-subunits in the rat cardiomyocytes shows that the Cavß1b-GFP is primarily present in the T-tubules, which is similar to what we have detected in canine cardiomyocytes. ß2a-GFP (Cavß2aN3) is localized to the surface sarcolemma, whereas in our study the Cavß2 is primarily localized to the T-tubules with weak staining of the surface sarcolemma. This difference may reflect the use of the palmitoylated Cavß2aN3 fused with GFP in the prior study with its unique membrane targeting properties (11, 50), compared with the present study detecting endogenous Cavß2 isoforms which likely do not include Cavß2aN3 as described above. Colecraft et al. (13) show that both Cavß3-GFP and Cavß4-GFP are intracellular with predominant fluorescence in the nucleus. In contrast, we detect native Cavß3 and Cavß4 in the sarcolemmal membranes in the canine cardiomyocytes. There are limitations in overexpressing exogenous proteins which can complicate the interpretation of such studies. For example, strong overexpression of the channel subunits may interfere with the normal protein trafficking of these isoforms, and competition with endogenous subunits may also complicate the results.

The present study for the first time provides evidence for differential subcellular distribution of endogenous Cavß subunits in ventricular myocytes based on the combined results of membrane fractionation studies and immunocytochemistry. The Cavß1b, Cavß2, and Cavß3 isoforms are preferentially localized to the transverse tubules with a weaker presence in the surface sarcolemma. Conversely, the Cavß1a and Cavß4 are preferentially localized to the surface sarcolemma and markedly less signal in the T-tubules. Regardless of the Cavß subcellular targeting, we hypothesize that the majority of Cavß isoforms colocalize with Cav1.2 subunits either at the surface membrane or in the T-tubules, but we did not do coimmunostaining experiments to rigorously verify this. Given the prominent role of L-type Ca2+ channels in the T-tubules in initiating excitation-contraction coupling, it is possible that Cavß1b, Cavß2, and Cavß3 subunits importantly contribute to excitation-contraction coupling. Whether the Cavß1a and Cavß4 subunits contribute to alternative cell processes, such as cellular signaling, remains to be determined. Unfortunately, specific antibodies are only available for a minority of the Cavß isoforms detected at the message level in this study, and so further refinement of the subcellular localization of many individual splice variants will require future study.

It is intriguing that two splice variants from the same Cavß1 gene are localized differentially. Cavß1a localizes primarily to the surface sarcolemma, whereas Cavß1b preferentially targets to the transverse tubule sarcolemma. There are two main differences in these isoforms with differential splicing of the exons 7A (Cavß1a) and 7B (Cavß1b), as well as an additional exon 14 present only in Cavß1b. The functional importance of alternatively spliced exons 7A and 7B may be connected to their close proximity to the "beta interaction domain" (BID) in the adjacent exon 8. Studies have shown that all known Cavß subunits interact with Cav1 and Cav2 subunits through a high-affinity interaction site (BID) localized to a 30-amino acid region at the beginning of the D4 domain of the ß-subunit (16). Thus alternative exons in this general region may alter the interactions with the {alpha}-subunits and potentially affect membrane trafficking. Alternatively, the distinct COOH termini of Cavß1a and Cavß1b may make for different subcellular targeting. Differential interactions of the Cavß1 isoforms with cellular proteins other than the Cav{alpha} subunit likely play a major role in membrane trafficking and localization. The interaction with other proteins seems particularly possible for the Cavß1 subunits given the proline-rich amino terminus homologous to a PDZ domain with an overall structure typical of the membrane-associated guanylate kinase (MAGUK) protein (27). The MAGUK protein family includes proteins such as PSD95 and can be important in targeting ion channels and membrane proteins (37). In addition, members of the RGK family of GTPases have recently been identified as interacting directly with Cavß subunits, and these proteins have been shown to dramatically impact membrane trafficking of the channel complex (3, 19).

Functional impact of Cavß structural diversity.
The remarkable diversity of the Cavß subunits in the heart begs the question of the functional impact and cellular roles played by these distinct subunits. It has long been clear that ß-subunit isoforms differ in their functional effects, as studied in heterologous expression systems and more recently in cardiomyocytes (13, 55). Many studies have described multiple functional effects of coexpression of Cavß subunits with pore-forming Cav{alpha} subunits. These modulatory effects are limited to the Cav1 and Cav2 families of {alpha}1-subunits, not the low-voltage-activated Cav3 family. There are four general categories of effects: 1) changes in channel gating; 2) alterations in membrane trafficking and localization of channels; 3) regulation of channels by second messenger systems; and 4) alterations in drug block properties. For example, Cavß subunit coexpression can shift the voltage dependence of channel activation and inactivation substantially, and these shifts vary in direction and magnitude depending on the Cavß subunit studied. Another potential important difference between Cavß subunits exists for the regulation of Cav1.2 channels by protein kinase A (PKA) that, in part, involves the specific phosphorylation of residues uniquely found in the carboxy terminus of the Cavß2 subunit and not the other Cavß subunits (23). Thus the different functional capabilities of each of the Cavß subunit isoforms may allow for highly specific modulation of the L-type Ca2+ channel complex.

In the present study, we focused on exploring the functional properties of three novel splice variants Cavß2cN1, Cavß2cN2, and Cavß2cN4, which we cloned from human heart and vary only in the amino terminus. The Cavß2c subunits have previously been suggested, in part, at the message level by detection of alternative exon 7C (13, 52), but they have not previously been cloned in full-length or functionally characterized. When the Cavß2c subunits were coexpressed with Cav1.2 and Cav{alpha}2{delta} in HEK 293 cells, we noted a large increase in expressed current compared with Cav1.2 + Cav{alpha}2{delta} channels that was likely due to both changes in channel gating and membrane trafficking of channel complexes as previously described for full-length Cavß subunits (5, 12, 24, 46). Furthermore, differences in the precise modulation of gating by the Cavß2c subunits were detected based on the smaller negative shift in the voltage dependence of activation (V1/2) when Cavß2cN1 was coexpressed compared with Cavß2cN2 and Cavß2cN4. The Cavß2cN1 isoform is unique among these isoforms in that it includes the proline-rich amino terminus encoded by exon 1A. Similarly, a recent study has demonstrated distinct effects on channel activation by Cavß4bN1 and Cavß4bN2 (same isoforms detected in human and canine heart here) which were attributed specifically to the proline-rich region in Cavß4bN1 (30). In contrast, all three Cavß2c isoforms accelerated channel inactivation similar to the Cavß2aN4.

Takahashi et al. (57) recently studied all five NH2-terminal splice variants of Cavß2 in the Cavß2a backbone and likewise revealed NH2-terminal splice variant-specific effects on channel gating. The Cavß2cN1, Cavß2cN2, and Cavß2cN4 splice variants that we evaluated all accelerated inactivation similarly to the matched subunits in the previous study: ß2d (Cavß2aN1), ß2c (Cavß2aN2), and ß2b (Cavß2aN4). In comparison, Takahashi et al. (57) found that ß2a (Cavß2aN3) and ß2e (Cavß2aN5) resulted in a relative slowing of inactivation, but we did not test these isoforms. The variations in voltage-dependent activation observed by Takahashi et al. (57) with the Cavß2a amino terminal splice variants did not simply match with the shifts in voltage-dependent activation that we observed for the Cavß2c isoforms, suggesting that the aggregate structure of both the amino terminus and the central variable region modulate activation. These recent results add to earlier work demonstrating the critical role of the amino terminus and central domain of the Cavß subunit in finely regulating channel gating (45, 49).

Perhaps the most unique and functionally distinct family of Cavß subunit isoforms will be the "d" isoforms, including the Cavß1d, Cavß2dN4, ß4dN1, and ß4dN2 isoforms identified in this report. As these isoforms skip exon 7, a frame shift results in a stop codon and the predicted protein is truncated, lacking the BID and the entire carboxy half of the protein present in other Cavß isoforms. Recent studies have identified a ß4c in the chick cochlea in agreement with our finding of a related isoform in human and canine heart. This protein was shown to be multifunctional in that it could not only modulate channel gating but also could target to the nucleus and regulate gene transcription (31). Another recent study has shown that ß3trunc (Cavß3d, not detected in the heart in our study) expression is increased in human ischemic cardiomyopathy, and expression with Cav1.2 and Cav{alpha}2{delta} in heterologous expression systems alters the open probability (Po) of the expressed L-type Ca2+ channel (33). Thus the "d" isoforms can still apparently interact with the channel but also may, in some cases, have important gene regulation functions.

Alternative splicing in the COOH terminus of Cavß subunits may also have functional impact. Splicing of the COOH terminus is only well described for the Cavß1 gene. In this case, the functional impact is most clearly seen in the essential role of the COOH terminus of Cavß1a in voltage-dependent excitation-contraction coupling of skeletal muscle (4, 54). Whether the different COOH termini of the Cavß1 subunits impact excitation-contraction coupling in cardiac muscle is unknown.

Overall, the remarkable diversity of Cavß subunits expressed in the heart demonstrates the presence of multiple functionally distinct populations of L-type Ca2+ channels. The modular nature of the Cavß subunit provides for alternative splicing that can precisely regulate channel gating, regulation, and localization. Thus subpopulations of Ca2+ channels may subserve distinct cellular functions. In addition, Cavß subunits may participate in other cell processes such as gene regulation independent of the channel complex. We are only beginning to appreciate the roles of Cavß subunits in cell biology.


   GRANTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Institutes of Health Grants R01-HL-61537 and P01-HL-47053 (to T. J. Kamp), R01-HL-60723 (to C. T. January), and NRSA Postdoctoral Fellowship Award F32-HL-071476 (to B. P. Delisle).


   ACKNOWLEDGMENTS
 
The assistance of Thankful Sanftleben with manuscript preparation and of Jo Ellen Lomax with molecular biology is gratefully acknowledged. We also thank Kevin P. Campbell for providing the Cavß1a antibody.


   FOOTNOTES
 
Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).

Address for reprint requests and other correspondence: T. J. Kamp, H6/343 Clinical Science Center, Box 3248, 600 Highland Ave., Madison, WI 53792 (E-mail: tjk{at}medicine.wisc.edu).

10.1152/ physiolgenomics.00207.2003.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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