Alternative Splicing in Intracellular Loop Connecting Domains II and III of the alpha 1 Subunit of Cav1.2 Ca2+ Channels Predicts Two-domain Polypeptides with Unique C-terminal Tails*

Paul A. Wielowieyski, Jeffrey T. Wigle, Maysoon Salih, Peggy Hum, and Balwant S. TuanaDagger

From the Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, Ontario K1H 8M5, Canada

Received for publication, July 31, 2000, and in revised form, September 27, 2000



    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Novel splice variants of the alpha 1 subunit of the Cav1.2 voltage-gated Ca2+ channel were identified that predicted two truncated forms of the alpha 1 subunit comprising domains I and II generated by alternative splicing in the intracellular loop region linking domains II and III. In rabbit heart splice variant 1 (RH-1), exon 19 was deleted, which resulted in a reading frameshift of exon 20 with a premature termination codon and a novel 19-amino acid carboxyl-terminal tail. In the RH-2 variant, exons 17 and 18 were deleted, leading to a reading frameshift of exons 19 and 20 with a premature stop codon and a novel 62-amino acid carboxyl-terminal tail. RNase protection assays with RH-1 and RH-2 cRNA probes confirmed the expression in cardiac and neuronal tissue but not skeletal muscle. The deduced amino acid sequence from full-length cDNAs encoding the two variants predicted polypeptides of 99.0 and 99.2 kDa, which constituted domains I and II of the alpha 1 subunit of the Cav1.2 channel. Antipeptide antibodies directed to sequences in the second intracellular loop between domains II and III identified the 240-kDa Cav1.2 subunit in sarcolemmal and heavy sarcoplasmic reticulum (HSR) membranes and a 99-kDa polypeptide in the HSR. An antipeptide antibody raised against unique sequences in the RH-2 variant also identified a 99-kDa polypeptide in the HSR. These data reveal the expression of additional Ca2+ channel structural units generated by alternative splicing of the Cav1.2 gene.



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The voltage-gated ion channels determine membrane excitability and regulate signal transduction (1-5). These ion channels consist of multimeric complexes comprising a central alpha  subunit, which contains the structural determinants for ion selectivity, conductance, and voltage sensing, and several auxiliary subunits, which confer regulation on the alpha  subunits (6, 7). The alpha  subunits of K+, Na+, and Ca2+ channels show overall structural similarity in that the alpha  subunit of K+ channels consists of a single domain, which is predicted to contain six membrane-spanning (S1-S6)1 regions, whereas the structural unit of Na+ and Ca2+ channels consists of four such homologous domains (1). Because the K+ channel genes encode only one domain, it is proposed that homo- and heterotetrameric co-assembly of single-domain K+ channel subunits is required to constitute the four-domain channel complexes (8-10).

The different pore-forming alpha  subunits are encoded by distinct gene families (68). Ten genes encoding the voltage-gated Ca2+ channels have been identified; seven (denoted Cav1.1-1.4 and Cav2.1-2.3) encode the high voltage-activated channels (5, 11-17), and three (Cav3.1-3.3) encode the low voltage-activated channels (18-21). Genes encoding different alpha 1 subunits of the voltage-gated Ca2+ channels exhibit distinct channel properties, which are determined by subtle changes in amino acid composition and appear to be expressed in a tissue- and cell-specific manner (22-28). Alternative splicing, posttranslational modifications, and modulation by auxiliary subunits can generate further diversity in Ca2+ channel heterogeneity, although the four-domain structure is maintained (29-36). For example, alternative splicing of mutually exclusive exons encoding the S3 segment of domain IV of the alpha 1 subunit of Cav1.2 channels serves as a developmentally regulated switch in cardiac tissue coinciding with major changes in excitation (29). Variability in the carboxyl-terminal region of the alpha 1 subunit of Cav1.2 channels generated by alternative splicing influences the kinetics as well as Ca2+ and voltage dependence of the L-type Ca2+ channels (32, 33). Moreover, dihydropyridine sensitivity of cardiac and vascular alpha 1 subunits of Cav1.2 Ca2+channels may be attributed to tissue-specific expression of an alternatively spliced S6 segment of domain I of the Cav1.2 gene (36).

Changes in amino acid composition in the extramembrane regions of the alpha 1 subunit appear to have given rise to specialized physiological roles for the different classes of alpha 1 subunits of the Ca2+ channel family (37-41). For example, the structure of the intracellular loop connecting domains II and III appears to be a critical determinant of the mode of signal transmission in the Cav1.1 and Cav1.2 alpha 1 subunits (42-44), because this structure of the skeletal (Cav1.1) but not cardiac (Cav1.2) alpha 1 subunits can directly interact with the Ca2+ release channel of the sarcoplasmic reticulum to determine the contractile characteristics of skeletal muscle (45-47). Furthermore, loop II-III of Cav2.1 (P/Q-type) and Cav2.2 (N-type) Ca2+ channels associates with syntaxin and synaptosomal associated protein 25 to regulate excitation-secretion coupling (50-56).

Because the alpha 1 subunit of Cav1.2 Ca2+ channels is expressed in tissues such as the myocardium and brain, which are composed of a heterogeneous population of cells, it has been postulated that alternatively spliced variants may be expressed to serve functions in a cell type-specific manner. In view of the functional importance of the loop II-III structure, we examined whether alternative splicing will result in the generation and expression of structural variants in this critical region. We report here the identification of two splice variants of Cav1.2 Ca2+ channels that generate truncated forms of the alpha 1 subunits, which were predicted to constitute domains I and II with unique carboxyl-terminal tails. The expression of these variants in cardiac and neuronal tissue suggests that further diversity in Ca2+ channel function and regulation may be generated through alternative structural units.


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Isolation of RNA and RT-PCR Analysis-- Total RNA was isolated using the TriPure reagent (Roche Molecular Biochemicals) according to method described by Chomczynski (48). Adult rabbit heart, brain, and skeletal muscle were frozen in liquid nitrogen, and total RNA was extracted in phenol thiocyanate. Total RNA (1.0 µg) was reverse transcribed with either a gene-specific primer, based on the sequence of the alpha 1 subunit of the Cav1.2 gene from rabbit myocardium described by Mikami et al. (Ref. 17; primer JW6, GGTGAAGATCGTGTCGTTGAC, nt 2990-2970) or random hexamers and SuperScript reverse transcriptase. The first-strand cDNA synthesis with the gene-specific primer results in exclusive reverse transcription of the Cav1.2 transcripts from rabbit myocardium, thus yielding increased sensitivity of the PCR amplification by increasing the representation of the rare messages. To amplify the Cav1.2 subunit loop II-III, the specific primers JW5 (GTGGACAACCTGGCTGATGCTGAG, nt 2538-2561) and JW6 were used. The PCR was performed under the following conditions: 94 °C for 45 s, 50 °C for 30 s, and 72 °C for 30 s, repeated 30 times. There were two negative controls used, one with water to control for contamination of the master mix and a second control reaction that contained all reaction components except reverse transcriptase (i.e. RNA but no first-strand cDNA) to control for contamination from genomic DNA and partially processed mRNA. The PCR reaction was run in an MJ Research thermocycler. The products were analyzed by 1% gel electrophoresis, gel purified, and cloned into the pCRII vector (Invitrogen). Twenty bacterial colonies were selected and screened by restriction mapping.

Amplification of the loop II-III variant fragments encompassing the predicted stop codon at nucleotide 2995 of the Cav1.2 gene used upstream and downstream primers anchored in transmembrane sequences encoding domains II and III of cardiac alpha 1 subunit. The sense primer loop5' (TCCTACTGAATGTGTTGG, nt 2509-2529) and antisense primer loop3' (CAGGATG TTGAAGTAGTTC, nt 3199-3179) also incorporated BamHI and EcoRI restriction sites, respectively, to facilitate directional cloning of the PCR products into pTZ18 (United States Biochemical, Cleveland, OH). Similar to RT-PCR performed with JW5 and JW6 primers, synthesis of the first-strand cDNA was primed with a gene-specific antisense loop3' oligonucleotide. The following cycling condition were used: 94 °C for 45 s, 52 °C for 35 s, and 72 °C for 35 s, repeated 32 times. The final elongation was performed at 72 °C for 10 min. The RT-PCR products were analyzed on a 1% agarose gel, gel purified, and subcloned into the pTZ18 vector. Sixty bacterial colonies were selected and screened by restriction mapping and direct sequencing.

Screening of Recombinant Clones and Cycle Sequencing-- Recombinant clones of loop II-III RT-PCR amplicons subcloned into either pCRII or pTZ18 were selected and inoculated overnight at 37 °C in LB broth containing 75 µg/ml ampicillin. Plasmid DNA was isolated using an alkaline miniprep (49). The recombinant constructs were screened by restriction enzyme digestion and agarose gel electrophoresis to identify loop II-III deletion variants. The various inserts were then purified with a Qiagen miniprep plasmid preparation method to obtain high-purity templates suitable for cycle sequencing with the Applied Biosystems Prism dye terminator cycle-sequencing method (PerkinElmer Life Sciences), and the sequences were analyzed using SeqAidII tools (University of Kansas).

Cloning of Full-length alpha 1 Subunit Variant cDNAs-- Total RNA (1.0 µg) prepared as described above was reverse transcribed with antisense primer 5'-CAGGATGTTGAAGTAGTTC-3' (nt 3199-3179) and SuperScript at 50 °C for 45 min. PCR amplification was carried out at standard conditions with sense primer 5'-TGGAAACTGACAATGCTTCGAGCC-3' (nt 180-203) and 3' antisense splice-specific primers 5'-ATCCTCTTCTCCTTGGCCTCCTC-3' and 5'-GAGGCGGAACCTGTGGTTTCC-3', with first denaturation at 94 °C for 2 min followed by 94 °C for 45 s, 70 °C for 35 s, 72 °C 2 min 30 s, which was repeated 32 times, and the final elongating at 72 °C for 10 min. The PCR amplicons were electrophoresed on a 0.9% agarose gel, TA cloned into pCRII vector (Invitrogen), and cycle sequenced (PerkinElmer Life Sciences).

RNase Protection Assay-- The RNase protection assay used two cRNA probes, RH-1 and RH-2, representing deletion variants of loop II-III lacking exon 19 and exons 17 and 18. The 32P-radiolabeled deletion variant probes were generated by in vitro transcription using MAXIscript reagents (Ambion) with [alpha -32P]UTP (PerkinElmer Life Sciences); 104 cpm of the respective RNA probe was hybridized with 15 µg of mRNA in a Hybspeed hybridization at 68 °C for 10 min followed by RNase digestion (RNase A/T1, 1:100) at 37 °C for 45 min. Reactions were inactivated by addition of inactivation-precipitation mix (Ambion) and ethanol precipitated. Pellets were resuspended in 10 µl of gel loading buffer and analyzed on 5% polyacrylamide-8 M urea-1× Tris borate-EDTA vertical gel and subjected to autoradiography. The protected fragments appearing on the autoradiograms were quantified on a Kodak Science image analysis station. To assess relative expression of Cav1.2 loop II-III variants, the intensities of protected fragments were size normalized and expressed as a ratio. Century RNA markers (Ambion) were used as molecular size standards.

Antibodies, Western blot Analysis, and Subcellular Fractionation of Cardiac Membranes-- Affinity-purified antibodies directed against peptide sequences (KYTTKINMDDLQPSENEDKS) of the intracellular loop II-II of the Cav1.2 alpha 1 subunit were generously provided by Dr. William Catterall. Polyclonal antibodies directed against the carboxyl-terminal sequences of RH-1 and RH-2 variants were raised in New Zealand White rabbits. The only unique amino acid sequence (TTGSASSVTVSSTTRSSPT) of the common 19-amino acid carboxyl terminus of RH-1 and RH-2 was found to be nonantigenic and gave no immune response. On the other hand, two peptide sequences, KRMRRSLRCLSAPALGHS and GHSPSCTLRRRPC, were selected to raise antibody 1929 selective for the RH-2 variant. The peptides were conjugated to keyhole limpet hemocyanin carrier protein via the carboxyl-terminal carboxyl group and used in six consecutive immunizations per each of the New Zealand White rabbits (Genosys). The antisera was collected in four bleeds per animal. The Na+/K+-ATPase antibodies were provided by Dr Kathleen Sweadner.

Membrane fractions from rat myocardium were isolated by differential and sucrose density gradient centrifugation and characterized for the enrichment of markers for the sarcolemma (~20-fold enrichment in Na+/K+-ATPase and adenylate cyclase activities) and heavy sarcoplasmic reticulum (~10-fold enrichment in ryanodine receptors) as described (67). Membrane proteins were subjected to SDS-polyacrylamide gel electrophoresis followed by transfer to nitrocellulose membranes, which were probed with affinity-purified antipeptide antibodies.


    RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Identification of Two Alternatively Spliced loop II-III Variants of Cav1.2 Ca2+ Channels-- The second intracellular loop connecting domains II and III of the alpha 1 subunit of voltage-gated Ca2+ channels is believed to reside at the cytoplasmic phase of the membrane and determines the mode of signal transduction in excitation-contraction coupling in muscle cells (42-47) and excitation-secretion coupling in nerve cells (50-56). Given the structural and functional importance of this region, we determined whether diversity in Ca2+ channel structure can be created through alternative splicing in this region of the alpha 1 subunit gene. The intracellular loop connecting domains II and III of Cav1.2 alpha 1 subunits was amplified from rabbit myocardium by RT-PCR using primers based on the cDNA sequence reported by Mikami et al. (17). The first-strand cDNA synthesis was primed either with the Cav1.2 subunit-specific oligonucleotide JW6 (nt 2990-2970) or random hexamers. The purpose of priming with the gene-specific oligonucleotide was to increase representation of the rare messages of the Cav1.2 transcript family. The PCR amplification of loop II-III was then carried out with JW5 (nt 2538-2561) and JW6 (nt 2990-2970) primers and analyzed by 1% agarose electrophoresis (Fig. 1A). The PCR with the JW6 primer yielded a major 452-nt product as well as smaller diffuse fragments (Fig. 1A, lane 1), whereas PCR of the first-strand cDNA primed with random hexamers yielded a single fragment of 452 nt (Fig. 1A, lane 3). Fig. 1A, lane 2, represents a PCR-negative control. Subcloning and direct sequencing of the PCR products shown in lane 1 revealed that the 452-nt product matched perfectly the sequence of the intracellular loop linking domains II and III of the rabbit heart alpha 1 Cav1.2 subunit gene, whereas that of the smaller diffuse fragment matched the 452-nt loop II-III sequence except for a deletion of 133 nt (from 2812 to 2945). Open reading frame analysis of the 320-nt amplicon containing the 133-nt deletion indicated that the deletion was predicted to shift the translational reading frame of full-length alpha 1 Cav1.2 and to introduce a premature chain termination at nt 2995 (data not shown). To obtain longer cDNAs encompassing the entire loop II-III and the predicted stop codon, RT-PCR was performed with another set of primers nested in the transmembrane segment S6 of domain II and S3 segment of domain III (loop 5', nt 2509-2529; and loop 3', nt 3199-3179, respectively). Primers were designed to contain BamHI and EcoRI restriction enzyme sites to facilitate subcloning and analysis of the PCR amplicons. Fig. 1B depicts RT-PCR of rabbit heart, where the first-strand cDNA was performed with the gene-specific antisense oligonucleotide loop 3', and PCR amplification was carried out with loop 5' and loop 3' primers. Similar to RT-PCR analysis carried out with the JW5 and JW6 primers, amplification with the loop 5' and loop 3' primers produced two products, an amplicon of ~700 nt and a lower band of ~560 bp (Fig. 1B, lane 1). Moreover, the size difference between the top and bottom products was ~ 140 nt, analogous to the RT-PCR performed with the JW5 and JW6 primers. After subcloning of the amplified products, three representative amplicons were characterized by restriction mapping and direct sequencing (Fig. 1C). Lane 1 shows the presence of a 690-nt product, representing the expected amplification product of alpha 1 Cav1.2 with the loop 5' and loop 3' primers, and lanes 2 and 3 represent two clones isolated and characterized from the lower RT-PCR amplification band. The nucleotide sequence analysis and alignment shown in Fig. 2A indicated that the 690-nt product represented the nucleotide sequence of the expected amplified fragment encompassing the wild-type intracellular loop II-III of Cav1.2 (Rab-H), whereas the smaller products denoted two distinct variants of this sequence. Variant 1, denoted RH-1, and variant 2, denoted RH-2, appear to be derived from the full-length loop II-III sequence spanned by the loop 5' and loop 3' primers, as a consequence of internal deletions of 133 and 130 nt, respectively. The RH-1 variant contained deletion of nt 2812-2945, and it represents a deletion variant already characterized in the RT-PCR analysis carried out with the JW5 and JW6 primers. The RH-2 variant displayed deletions of nt 2681-2811, and it represents a novel variant cloned from the smaller amplification product of loop II-III. Analysis of the open reading frame of the deletion variants RH-1 and RH-2 in relation to the wild-type sequence confirmed that both deletions shifted the open reading frame, thus introducing a common termination codon (TGA) at nucleotide 2995. The amino acid sequence alignment of the variants indicated that both variants introduce novel amino acid sequences past their respective deletions depicting novel carboxyl terminal sequences of the truncated polypeptides (Fig. 2B). Because both variants use the same stop codon, the terminal 19 amino acids were identical in the two variants. An independent isolation of the RH-1 and RH-2 cDNAs was obtained with RT-PCR of RNA from the whole heart and cardiac left ventricle but not skeletal muscle.



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Fig. 1.   RT-PCR of loop II-III of the cardiac alpha 1 subunit. A, loop II-III of the rabbit cardiac alpha 1 Cav1.2 subunit of the L-type Ca2+ channel was amplified by RT-PCR with JW5 and JW6 primers. The RT reaction was primed with the antisense JW6 oligonucleotide (lane 1) or with random hexamers (lane 3). A control reaction containing no first-strand cDNA template (i.e. no reverse transcriptase) is represented in lane 2. The amplification of the predicted 452-nt product is seen in lanes 1 and 3. In addition, lane 1 also indicates the presence of smaller products of ~300 bp. B, results of the RT-PCR analysis of the rabbit cardiac alpha 1 Cav1.2 with loop 5' and loop 3' primers, encompassing loop II-III and the predicted stop codon at nt 2995. The analysis reveals the presence of two amplification products, similar to the RT-PCR shown in A, thus confirming the expression of deletion variants of loop II-III. C, EcoRI-BamHI digests of representative recombinant plasmids derived by cloning DNA fragments eluted from a gel in which the RT-PCR products from cardiac RNA were amplified with primers anchored in the membrane-spanning segments S6 of domain II (loop 5', nt 2509-2529) and S3 of domain III (loop 3', nt 3199-3179). The products from the RT-PCR reaction were separated by gel electrophoresis, and the amplification products were subcloned as described under "Experimental Procedures." Three representative clones were characterized by the restriction mapping and direct sequencing (lanes 1-3).



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Fig. 2.   Characterization of PCR products. A, DNA sequence analysis and alignment of the loop II-III variants. Shown are the nucleotide sequence and alignment of the various RT-PCR products of the alpha 1 subunit loop II-III amplified with loop 5' and loop 3' primers from Fig. 1B. RH-1 and RH-2 denote the two smaller fragments; Rab-H represents the larger product in Fig. 1B. The sequence and alignment reveal that RH-1 and RH-2 contain deletions of 133 and 130 nt, respectively, when compared with the sequence of the full-length (wild-type) loop II-III (Rab-H). The sequence gaps are denoted by dashes, and the deduced in-frame stop codons are shaded. The splice junction primers are single or double underlined. All sequence positions are relative to the rabbit cardiac alpha 1 subunit (X15539) identified by Mikami et al. (43). B, amino acid alignment of the loop II-III variants. Shown is alignment of the deduced amino acid sequence of the loop II-III splice variants, where boldface denotes the novel carboxyl terminus common to RH-1 and RH-2 splice variants, and the underlined sequence represents the unique RH-2 sequence. C, genomic organization of the loop II-III variants. A schematic representation of the loop II-III splice variants based on the exon organization of intact loop II-III of the cardiac alpha 1 subunit gene is indicated. Hum-H represents the full-length (wild-type) loop II---III; RH-1 and RH-2 represent the two deletion variants. It is depicted that the RH-1 variant would arise because of deletion of exon 19, and as a result there would be a reading frameshift of exons 20. The RH-2 variant, on the other hand, would be missing a fragment of exon 17 and the entire exon 18; this would also result in a reading frameshift of exons 19 and 20. The reading frameshifts in both variants would introduce a common premature termination codon in exon 20.

Evaluation of Genomic Organization of the Loop II-III Variants-- The comparison of the deletion sites of the loop II-III variants with the exon-exon boundaries of the human alpha 1 Cav1.2 subunit gene is summarized (Fig. 2C). Depicted are the exons encoding loop II-III of the Cav1.2 alpha 1 subunit gene as determined by Soldatov (57) and how the variants would potentially arise as a result of internal deletions. The RH-1 variant lacks exon 19, which results in a reading frameshift of exon 20, thus generating a novel 19-amino acid carboxyl-terminal peptide with an in-frame premature termination codon. The RH-2 variant, on the other hand, could arise because of to the deletion of a 3' 60-nt fragment of exon 17 and the entire exon 18. The putative alternative 5' donor site of the variant would be in exon 17, whereas the 3' splice site of exon 19 would be used as a 3' acceptor. The deletion of the 3' part of exon 17 and the entire exon 18 predicts a reading frameshift, such that exons 19 and 20 are translated with respect to a new reading frame. Consequently, exons 19 and 20 are predicted to translate a novel 62-amino acid carboxyl-terminal polypeptide.

mRNA Expression of Cav1.2 Ca2+ Channel Variants-- Confirmation of the expression of the Cav1.2 subunit mRNA variants detected by RT-PCR was sought by RNase protection assays. Fig. 3 represents a schematic of the predicated sizes of the protected fragments that would be expected with the RH-1 and RH-2 probes. An antisense RNA probe of RH-1 was constructed based on the 323-nt amplicon of loop II-III (exons 16-18 and 20). The mRNA from cardiac and fast twitch skeletal muscle was used to protect the RH-1 probe, and a representative autoradiogram is shown. The mRNA from cardiac muscle protected the 323-nt probe representing the RH-1 variant as well as fragments of ~274, 144, and 46 nt (Fig. 3, lane 3). The 323-nt protected fragment represents the RH-1 type variant of loop II-III and would correspond to a part of exon 16 through to the end of exon 18 and the beginning of exon 20. Exon 19 of 133 nt is deleted in the RH-1 splice variant. The 274-nt protected fragment denotes a part the of the wild-type alpha 1 subunit loop II-III, from exons 16 to 18. Because exon 19 is spliced out in the RH-1 type variant, this exon is unprotected and consequently degraded by RNase. The theoretically expected fragments of 144 and 46 nt, which would be contributed by the RH-2 variant and the 3' end of the wild-type loop II---III, respectively, were detectable, although at low levels, perhaps reflecting low specific activity of the probe and the sensitivity of the assay as well as the expression levels. A fragment of 454 nt probably represents trace amounts of undigested cDNA template that co-precipitated with RH-1 cRNA probe as it migrates at the same level as the free undigested probe (Fig. 3, lane1). RNA from skeletal muscle failed to protect any fragments (Fig. 3, lane 4). The protection of multiple fragments by RNA from cardiac muscle is suggestive of the expression of the RH-1-type alternatively spliced transcript and the wild type loop II-III of the Cav1.2 gene.



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Fig. 3.   RNase protection analysis of loop II-III. Expression analysis of loop II-III variant mRNAs was performed with an RNase protection assay using RH-1 and RH-2 cRNA probes. A schematic depicts the expected products of the RNase protection assay of the alpha 1 subunit of Cav1.2 Ca2+ channels with the deletion variant probes isolated by RT-PCR. The line diagram reveals the expected sizes of the protected fragments contributed by wild type (wt) and the deletion variants of the alpha 1 subunit loop II-III mRNA. Solid bars denote protected fragments; dotted lines represent deleted exons in RH-1 and RH-2 variants. Expected sizes of the protected fragments are indicated above the respective products. Bottom panel, results of the RNase protection of cardiac and skeletal muscles with RH-1 and RH-2 cRNA probes. The protection of the mRNA from rabbit heart (lane 3) and skeletal muscle (lane 4) with the RH-1 cRNA probe, lacking exon 19, is shown. Lane 1, migration of the free probe (open arrowhead); lane 2, its digestion with RNase. The RH-1 probe protected fragments of ~320, 274, 160, 144, and 46 nt in heart (solid arrows, lane 3) that closely corresponded to the sizes of the expected products. Interestingly, however, protection of an ~160-nt fragment (solid arrowhead) may perhaps be indicative of an additional deletion variant of loop II-III that has not yet been characterized. The RNase protection of mRNA from heart (lane 7) and skeletal muscle (lane 8) with the RH-2 type cRNA probe, lacking exons 17 and 18, is presented. Lane 5, migration of the free probe; lane 6, its digestion with RNase. The RH-2 probe protects fragments of ~320, 179, 144, and 46 nt, which closely correspond to the sizes of the expected fragments in the top panel.

An antisense RNA probe of RH-2 (320 nt) was constructed based on the RT-PCR amplicon of loop II-III (lacking exons 17 and 18). Protection of the RH-2 probe with cardiac and skeletal muscle mRNA was carried out, and the respective autoradiogram is shown (Fig. 3, lanes 5-8). The mRNA from cardiac muscle protected fragments of ~320, 179, 144, and 46 nt (Fig. 3, lane 7). The 320-nt protected fragment represents the RH-2 variant of loop II-III encoded by part of exons 16 and 17 and exons 19 and 20. The 179-nt protected fragment would be contributed by the protection of the 3' end of the wild-type loop II-III encoded by exons 19 and 20. Because the 3' 60 nt of exon 17 and the entire exon 18 are deleted in the RH-2 type variant, this region of loop II-III would not be protected and consequently degraded by RNase. The theoretically expected fragments of 144 and 46 nt contributed by the 5' end of the wild type loop II-III (exon 16 and the 5' end of exon 17) and the RH-1 deletion variant were also detected but at low levels. Skeletal muscle mRNA did not protect the RH-2 probe (Fig. 3, lane 8). The protection of multiple fragments by RNA from cardiac muscle is suggestive of the expression of the RH-1and RH-2 deletion variant transcripts as well as the wild type loop II-III. To estimate a relative level of expression of the three transcripts, the ratio of RH-1 and RH-2 deletion variants to the wild-type Cav1.2 loop II-III was quantified by using the relative band intensities of the RH-1 (320-bp band) and the RH-2 (323-bp band) protected fragments and the appropriate protected fragments contributed by the wild type loop II-III from the autoradiogram, 274 and 174 bp, respectively. After normalization, the analysis estimated a relative level of expression with a ratio 1:1.6:0.7 of wild-type loop II-III:RH-1:RH-2, respectively; however, the estimate remains to be confirmed with protein expression levels.

Brain mRNA also protected multiple fragments of RH-1 and RH-2 similar in sizes to those observed with cardiac mRNA (data not shown). Thus data from the RT-PCR amplification of the loop II-III splice variants together with the data from the RNase protection assays are indicative of the expression of these variants in cardiac and neuronal tissue but not skeletal muscle and are therefore consistent with the reported pattern of expression of the Cav1.2 alpha 1 subunit gene.

Domains I and II as Potential Structural-Functional Units of Ca2+ Channels-- On the basis of the identification of the loop II-III deletion variants, it was hypothesized that the alternatively spliced forms of alpha 1 subunit of RH-1 and RH-2 would generate two-domain truncated forms of the Cav1.2 Ca2+ channels. To verify the hypothesis, the full-length cDNAs encoding two-domain polypeptides needed to be selectively isolated. RT-PCR of cardiac RNA was performed with a common 5' sense primer (nt 180-203, TGGAAACTGACAAT GCTTCGAGCC) and the respective splice junction primers selective for the RH-1 and RH-2 variants (ATCCTCTTCTCCTTGGCCTCCTC-3' and 5'-GAGGCGGAACCTGTG GTTTCC, respectively). RT-PCR yielded two products of ~2.6 (Fig. 4, lane 1) and 2.4 (Fig. 4, lane 2) kb, representing RH-1 and RH-2 variants, respectively, which were subsequently TA cloned and then cycle sequenced. Lane 3 represents a PCR-negative (water) control. A control with RNA but without RT (for contamination with genomic and partially processed mRNA) was also run, and it was negative.



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Fig. 4.   RT-PCR of RH-1 and RH-2 deletion variants. To amplify the full-length two-domain channels RH-1 and RH-2, RT-PCR was performed using a common 5' primer (TGGAAACTGACAATGCTTCGAGCC) as well as splice junction-specific primers for RH-1 (ATCCTCTTCTCCTTGGCCTCCTC) and RH-2 variants (GAGGCGGAACCTGTGGTTTCC). Products of the RT-PCR analysis of rabbit cardiac RNA electrophoresed on a 0.9% agarose gel stained with ethidium bromide are shown. Lane 1, RH-1 amplicon of ~2.6 kb generated with the 5' primer and an RH-1-specific primer; lane 2, RH-2 variant of ~2.4 kb generated with the 5' primer and an RH-2-specific primer; lane 3, negative PCR (water) control. Products were gel extracted, TA subcloned, and sequenced.

The nucleotide sequence and the deduced amino acid sequence analysis of the RH-1 and RH-2 variants are shown in Fig. 5, which indicates identical nucleotide and amino acid sequences in domains I and II when aligned with the wild-type Cav1.2 alpha 1 subunit. The salient sequence differences were present in loop II-III as indicated The RH-1 variant would generate a polypeptide of predicted molecular mass of 99.0 kDa, whereas the RH-2 variant would encode a polypeptide of 99.2 kDa.



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Fig. 5.   RH-1 and RH-2 splice variants encode two-domain Cav1.2 Ca2+ channels. Sequence comparison of the full-length deletion variants of RH-1 and RH-2 type with alpha 1 subunit of the Cav1.2 Ca2+ channel are shown. Truncated channel variants contain identical sequences in domains I and II similar to those in alpha 1 Cav1.2, whereas the loop II-III sequences are divergent because of alternative splicing. The unique carboxyl-terminal sequences of truncated channels are enclosed in a black box. RH-1 and RH-2 variants encode two-domain alpha 1 Cav1.2 truncated channels of 99.0 and 99.2 kDa, respectively. Shaded sequences represent transmembrane segments based on predicted channel topology. The membrane-spanning regions (S1-S6) are shaded. ·, sequence identities; and , termination codons. The putative PKC phosphorylation sites in the carboxyl-terminal sequences of the deletion variants are denoted in bold and underlined. The RH-1 variant contains one putative PKC phosphorylation site, whereas RH-2 predicts three. The putative CK 2 phosphorylation site is represented in bold italics. The CK 2 putative site is not present in the deletion variants.

Confirmation of the in vivo expression of the two-domain Cav1.2 Ca2+ channel polypeptides was carried out by Western blot analysis of subcellular fractions from cardiac muscle with antipeptide antibodies specific for loop II-III (CNC-1) of the Cav1.2 calcium channel. The peptide sequence used as antigen is also preserved in the RH-1 and RH-2 variants. Subcellular fractions enriched in SL and HSR were immunoblotted with anti-Na+/K+-ATPase monoclonal antibody to confirm the enrichment of SL versus the SR (Fig. 6). The Na+/K+-ATPase staining is highly enriched in SL compared with the original microsomes and fraction HSR, and densitometery revealed that it was ~15-fold higher in SL compared with the other fractions. Immunoblotting with the affinity-purified CNC-1 antibody indicated the presence of an ~240-kDa polypeptide representing the alpha 1 Cav1.2 subunit in microsomes (Fig. 6, lane R) that was found to enrich in SL, although a substantial amount (~40%) of this immunoreactive polypeptide was also present in HSR. Interestingly, the CNC-1 antibody also recognized an ~99-kDa polypeptide in the microsomal fractions that was found to enrich in HSR but was completely absent from the sarcolemmal fraction. The immunoreactivity of CNC-1 with the ~240- and ~99-kDa polypeptides was completely negated by preincubation of the antibody with the antigen (data not shown). The 99-kDa peptide detected with the CNC-1 antibodies may represent the two-domain polypeptides generated by alternative splicing in loop II-III. To further confirm this, antibodies were raised against unique sequences in the RH-1 and RH-2 channel variants. The carboxyl-terminal 19 amino acids in RH-1 and RH-2 were used as the antigen; however, this peptide was found to be nonantigenic. Two peptide sequences unique to RH-2 (KRMRRSLRCLSAPALGHS and GHSPSCTLRRRPC) were found to be antigenic and used to obtain antiserum 1929. Fig. 6 demonstrates that antiserum 1929, selective for the RH-2 variant, immunoreacted with a 99-kDa polypeptide in the microsomal fractions as well as the HSR fractions but not the SL fraction. No reactivity was seen in any of the fractions with the preimmune serum. Thus the antibodies raised against the RH-2 variant identified a 99-kDa polypeptide that was absent from the SL fractions but was enriched in the heavy SR fraction as observed with the anti-Cav1.2 (CNC-1) antibody. These data suggest the presence of the two-domain Cav1.2 Ca2+ channel polypeptides generated by alternative splicing in loop II-III as components of the heavy SR membrane in cardiac muscle but not the SL.



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Fig. 6.   Subcellular distribution of Ca2+ channel polypeptides in myocardium. Crude microsomes from rat hearts were separated by differential and sucrose density gradient centrifugation to enrich for SL membranes and HSR and immunoblotted with specific antibodies to the Na+/K+-ATPase, anti-loop II-III sequence of the Cav1.2 Ca2+ channel (CNC-1) and Anti-RH-2 (1929). Immunoblotting with anti-Na+/K+-ATPase and the reactivity at ~110 kDa enriched in SL fractions compared with the crude microsomes (R). Immunoblotting with the anti-Cav1.2 Ca2+ channel (CNC-1) revealed reactivity with ~240- and ~99-kDa polypeptides in the various fractions. Immunoblotting with antisera 1929 raised against unique peptide sequences (KRMRRSLRCLSAPALGHS and GHSPSCTLRRRPC) of RH-2 indicated reactivity with a polypeptide of ~99 kDa in the various fractions with immune serum but not preimmune serum.

The predicted topology of the two-domain alpha 1 Cav1.2 subunit variants generated by alternative splicing from the proposed four-domain Ca2+ channel structure is shown (Fig. 7). The full-length RH-1 variant renders a truncated, two-domain polypeptide composed of normal domains I and II, as well as 78 amino acids of the wild-type loop and a novel 19-amino acid carboxyl-terminal tail. The RH-2 variant, on the other hand, contains normal domains I and II and 35 amino acids of the wild-type loop II-III and a unique 62-amino acid carboxyl-terminal peptide. The terminal 19 amino acids in both variants would be identical.



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Fig. 7.   Topology of full-length alpha 1 Cav1.2 and the deletion variants (RH-1 and RH-2). The topology of the full-length cardiac Cav1.2 Ca2+ channel and the predicted topology of the two-domain channels are shown. The RH-1 two-domain channel arises as a result of alternative splicing of exon 19, which results in reading frameshift of exon 20 and a premature termination codon. The RH-2-type channel, on the other hand, is generated by deletion of a fragment of exon 17 and entire exon 18, which results in a reading frameshift of exons 19 and 20. RH-1 and RH-2 both share a common 19-amino acid carboxyl terminus; however, RH-2 in addition contains a unique 43-amino acid sequence. The two-domain polypeptides retain the critical determinants of channel function, including activation, inactivation, voltage sensor, ion selectivity, and pore structure, which are contained within domains I and II. The beta -subunit binding sites will also be retained in the two variants, whereas the ligand binding sites for phenylalkylamines (PAA), the benzothiazepines (BTZ), and the dihydropyridines (DHP) will not be retained. The putative carboxyl-terminal PKC phosphorylation sites are indicated.

The intracellular loop of alpha 1 subunits connecting domains II and III of the Ca2+ channel family of proteins is a critical determinant of the mode of signal transduction through protein-protein interactions (45-47, 50-57), which can be further modulated by post-translational mechanisms such as phosphorylation of amino acid residues in this loop (51, 58-60). Although the consensus sequence prediction program PROSITE predicts putative PKC (TTK) and casin kinase 2 (TTGE) phosphorylation sites in the wild-type loop II-III, the demonstration of their phosphorylation and any functional consequences remains to be determined. Interestingly, however, the putative PKC site is deleted in the RH-2 variant, and the casin kinase 2 site is deleted on both RH-1 and RH-2, whereas the novel carboxyl termini introduce additional consensus PKC sites, one in RH-1 and three in RH-2. In view of the significant changes in the amino acid composition and perhaps tertiary structure of loop II-III in the two variants, it is speculated that these polypeptides (if functional) could markedly influence signal transduction via these structural units.

Although the role of the alpha 1 subunit variants producing truncated, two-domain Ca2+ channel polypeptides remains to be elucidated, existence of other two-domain channel polypeptides in the voltage-dependent superfamily has also been reported. A two-domain, truncated form of Cav1.1 alpha 1 subunits comprising domain I and chimeric domain IV generated by splicing of the S2 segment of domain II to the S2 segment of domain IV has been observed predominantly in muscle from neonates (61). In this regard, we observed that the RH-1 and RH-2 variants were expressed in fetal and neonatal hearts (data not shown). Genetic mutations of the neuronal Cav2.1 (P/Q-type Ca2+ channel, CACNL1A4), associated with inherited episodic ataxia, predict synthesis of a truncated, two-domain channel polypeptide composed of domains I and II and a part of domain III, suggesting that these molecules have functional consequences (62-64). In addition, a voltage-dependent Na+ channel alpha  subunit, SCN8A, generates a predicted polypeptide comprising domains I and II as a result of alternative splicing of exon 18N, which introduces an in-frame termination codon (65). The predicted topography of truncated CACNL1A4 is similar to that proposed for the SCN8A Na+ channel polypeptide. Recent studies revealed that the omega -conotoxin MVIIC receptor associated with Cav2.1 (P/Q-type) Ca2+ channels consisted of a 95-kDa polypeptide that comprised domains I and II and a part of loop II-III (66). The 95-kDa polypeptide was able to bind the auxiliary beta  subunits, and it remains to be determined whether the polypeptide can form functional channels. Furthermore, it would be intriguing to elucidate whether the 95-kDa polypeptide is also generated by the alternative splicing mechanisms of the Cav2.1 alpha 1 subunit gene, similar to the Cav1.2 gene. It is also notable that a unique cDNA has been isolated from kidney that encodes a two-domain polypeptide that exhibits ~20% identity with the voltage gated Na+ and Ca2+ channels (69).

The generation of two distinct polypeptides by alternative splicing is highly suggestive of functional specialization of the individual truncated alpha 1 subunit isoforms described here. The presence of the critical determinants of channel function, including activation and inactivation domains, the voltage sensor, ion selectivity, and pore structure within domains I and II, suggests that the truncated channel polypeptides could perhaps homodimerize and heterodimerize to form functional channels. The RH-1 and RH-2 variants may also play roles as dominant negative regulators of the wild type Cav1.2 Ca2+ channels. It is notable that subcellular distribution of the 240-kDa alpha 1 subunit indicated the presence of a substantial pool of this isoform in the heavy SR membranes, probably representing the dyad structure known to be enriched in this fraction. The exclusive presence of the 99-kDa RH-1 and RH-2 variants in this fraction suggests that these polypeptides may play a role in excitation-contraction coupling at the dyad junctions. In this regard, purified dyads were found to be highly enriched in both the 240- and 99-kDa polypeptides (data not shown). The RH-1 and RH-2 polypeptides may also serve regulatory roles by sequestering auxiliary channel subunits, such as the beta  subunit, because the binding site for this modulator would be retained by the two variants. In view of heterogeneity of the tissues in which the Cav1.2 alpha 1 subunits are expressed, it is conceivable that the splice variants may also be present in a subset of cells geared for specialized regulation of calcium entry. The level of expression of the two variants compared with the wild type is indicative of a stoichiometric relationship between the various isoforms. Studies indicate that there may be a voltage-dependent component to the excitation-contraction coupling mechanism in cardiac tissue (70), and whether the two domain polypeptides serve a role in this process remains to be defined.

In summary, the definition of the two new splice variants of the Cav1.2 alpha 1 subunit gene, which form two-domain polypeptides, implies a novel mechanisms for Ca2+ channel structure and function and/or regulation under normal physiological conditions. Moreover, these results lend further support for an evolutionary model of a four-domain channel structure created by two subsequent gene duplications of an ancestral one-domain gene.


    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Recipient of a grant from the Medical Research Council of Canada and Career Investigator of the Heart and Stroke Foundation of Ontario. To whom correspondence should be addressed: Dept. of Cellular and Molecular Medicine, University of Ottawa, 451 Smyth Rd., Ottawa, Ontario, Canada K1H 8H5. Phone: 613-562-5800 (ext. 8355); Fax: 613-562-5434.

Published, JBC Papers in Press, September 28, 2000, DOI 10.1074.jbc.M006868200


    ABBREVIATIONS

The abbreviations used are: S1-S6, membrane-spanning regions 1-6; loop II-III, second intracellular loop between domains II and III; RT-PCR, reverse transcription-polymerase chain reaction; RH, rabbit heart splice variant; nt, nucleotide; bp, base pair; SL, sarcolemma; SR, sarcoplasmic reticulum; HSR, heavy sarcoplasmic reticulum; PKC, protein kinase C; CNC-1, antipeptide antibody to loop II-III.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES


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