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• W2017492952 abstract "L-type Cav1.2 Ca2+ channel undergoes extensive alternative splicing, generating functionally different channels. Alternatively spliced Cav1.2 Ca2+ channels have been found to be expressed in a tissue-specific manner or under pathological conditions. To provide a more comprehensive understanding of alternative splicing in Cav1.2 channel, we systematically investigated the splicing patterns in the neonatal and adult rat hearts. The neonatal heart expresses a novel 104-bp exon 33L at the IVS3-4 linker that is generated by the use of an alternative acceptor site. Inclusion of exon 33L causes frameshift and C-terminal truncation. Whole-cell electrophysiological recordings of Cav1.233L channels expressed in HEK 293 cells did not detect any current. However, when co-expressed with wild type Cav1.2 channels, Cav1.233L channels reduced the current density and altered the electrophysiological properties of the wild type Cav1.2 channels. Interestingly, the truncated 3.5-domain Cav1.233L channels also yielded a dominant negative effect on Cav1.3 channels, but not on Cav3.2 channels, suggesting that Cavβ subunits is required for Cav1.233L regulation. A biochemical study provided evidence that Cav1.233L channels enhanced protein degradation of wild type channels via the ubiquitin-proteasome system. Although the physiological significance of the Cav1.233L channels in neonatal heart is still unknown, our report demonstrates the ability of this novel truncated channel to modulate the activity of the functional Cav1.2 channels. Moreover, the human Cav1.2 channel also contains exon 33L that is developmentally regulated in heart. Unexpectedly, human exon 33L has a one-nucleotide insertion that allowed in-frame translation of a full Cav1.2 channel. An electrophysiological study showed that human Cav1.233L channel is a functional channel but conducts Ca2+ ions at a much lower level. L-type Cav1.2 Ca2+ channel undergoes extensive alternative splicing, generating functionally different channels. Alternatively spliced Cav1.2 Ca2+ channels have been found to be expressed in a tissue-specific manner or under pathological conditions. To provide a more comprehensive understanding of alternative splicing in Cav1.2 channel, we systematically investigated the splicing patterns in the neonatal and adult rat hearts. The neonatal heart expresses a novel 104-bp exon 33L at the IVS3-4 linker that is generated by the use of an alternative acceptor site. Inclusion of exon 33L causes frameshift and C-terminal truncation. Whole-cell electrophysiological recordings of Cav1.233L channels expressed in HEK 293 cells did not detect any current. However, when co-expressed with wild type Cav1.2 channels, Cav1.233L channels reduced the current density and altered the electrophysiological properties of the wild type Cav1.2 channels. Interestingly, the truncated 3.5-domain Cav1.233L channels also yielded a dominant negative effect on Cav1.3 channels, but not on Cav3.2 channels, suggesting that Cavβ subunits is required for Cav1.233L regulation. A biochemical study provided evidence that Cav1.233L channels enhanced protein degradation of wild type channels via the ubiquitin-proteasome system. Although the physiological significance of the Cav1.233L channels in neonatal heart is still unknown, our report demonstrates the ability of this novel truncated channel to modulate the activity of the functional Cav1.2 channels. Moreover, the human Cav1.2 channel also contains exon 33L that is developmentally regulated in heart. Unexpectedly, human exon 33L has a one-nucleotide insertion that allowed in-frame translation of a full Cav1.2 channel. An electrophysiological study showed that human Cav1.233L channel is a functional channel but conducts Ca2+ ions at a much lower level. Voltage-gated calcium channels govern the depolarization-induced Ca2+ entry into cardiac muscles. The channel consists of a pore-forming α1 subunit associated with β, α2δ, and/or γ auxiliary subunits to form an oligomeric complex. In mammalian myocardium, excitation-contraction coupling is characterized by a transient increase in cytosolic Ca2+. The influx of Ca2+ through voltage-gated calcium channels subsequently induces Ca2+ release from sarcoplasmic reticulum in a process known as Ca2+-induced Ca2+ release. This process is prominent in the adult heart. The contraction of neonatal hearts relies more directly on the influx of Ca2+ through L-type voltage-gated calcium channels, in particular the Cav1.2 channels (1Vornanen M. Excitation-contraction coupling of the developing rat heart.Mol. Cell Biochem. 1996; 163: 5-11Crossref PubMed Scopus (23) Google Scholar, 2Vornanen M. Contribution of sarcolemmal calcium current to total cellular calcium in postnatally developing rat heart.Cardiovasc. Res. 1996; 32: 400-410Crossref PubMed Scopus (35) Google Scholar). L-type Cav1.2 channels are high voltage-activated channels and are expressed widely in cardiovascular and nervous systems (3Catterall W.A. Structure and regulation of voltage-gated Ca2+ channels.Annu. Rev. Cell Dev. Biol. 2000; 16: 521-555Crossref PubMed Scopus (1945) Google Scholar). The human Cav1.2 gene, CACNA1C, contains 55 exons, of which >19 exons are subjected to alternative splicing (4Liao P. Yong T.F. Liang M.C. Yue D.T. Soong T.W. Splicing for alternative structures of Cav1.2 Ca2+ channels in cardiac and smooth muscles.Cardiovasc. Res. 2005; 68: 197-203Crossref PubMed Scopus (102) Google Scholar). Novel splice variants of Cav1.2 channels were uncovered (5Cheng X. Liu J. Asuncion-Chin M. Blaskova E. Bannister J.P. Dopico A.M. Jaggar J.H. A novel Cav1.2 N terminus expressed in smooth muscle cells of resistance size arteries modifies channel regulation by auxiliary subunits.J. Biol. Chem. 2007; 282: 29211-29221Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 6Tiwari S. Zhang Y. Heller J. Abernethy D.R. Soldatov N.M. Atherosclerosis-related molecular alteration of the human Cav1.2 calcium channel α1C subunit.Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 17024-17029Crossref PubMed Scopus (72) Google Scholar) as of the first systemic screening of cardiac Cav1.2 channels (7Tang Z.Z. Liang M.C. Lu S. Yu D. Yu C.Y. Yue D.T. Soong T.W. Transcript scanning reveals novel and extensive splice variations in human l-type voltage-gated calcium channel, Cav1.2 α1 subunit.J. Biol. Chem. 2004; 279: 44335-44343Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). Alternative splicing of the Cav1.2 channels is coupled to the generation of splice variants with altered electrophysiological and pharmacological properties (4Liao P. Yong T.F. Liang M.C. Yue D.T. Soong T.W. Splicing for alternative structures of Cav1.2 Ca2+ channels in cardiac and smooth muscles.Cardiovasc. Res. 2005; 68: 197-203Crossref PubMed Scopus (102) Google Scholar, 8Liao P. Yu D. Li G. Yong T.F. Soon J.L. Chua Y.L. Soong T.W. A smooth muscle Cav1.2 calcium channel splice variant underlies hyperpolarized window current and enhanced state-dependent inhibition by nifedipine.J. Biol. Chem. 2007; 282: 35133-35142Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar9Liao P. Yu D. Lu S. Tang Z. Liang M.C. Zeng S. Lin W. Soong T.W. Smooth muscle-selective alternatively spliced exon generates functional variation in Cav1.2 calcium channels.J. Biol. Chem. 2004; 279: 50329-50335Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 10Abernethy D.R. Soldatov N.M. Structure-functional diversity of human L-type Ca2+ channel: perspectives for new pharmacological targets.J. Pharmacol. Exp. Ther. 2002; 300: 724-728Crossref PubMed Scopus (80) Google Scholar11Zühlke R.D. Bouron A. Soldatov N.M. Reuter H. Ca2+ channel sensitivity towards the blocker isradipine is affected by alternative splicing of the human α1C subunit gene.FEBS Lett. 1998; 427: 220-224Crossref PubMed Scopus (64) Google Scholar). They also show tissue-specific expressions (8Liao P. Yu D. Li G. Yong T.F. Soon J.L. Chua Y.L. Soong T.W. A smooth muscle Cav1.2 calcium channel splice variant underlies hyperpolarized window current and enhanced state-dependent inhibition by nifedipine.J. Biol. Chem. 2007; 282: 35133-35142Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 9Liao P. Yu D. Lu S. Tang Z. Liang M.C. Zeng S. Lin W. Soong T.W. Smooth muscle-selective alternatively spliced exon generates functional variation in Cav1.2 calcium channels.J. Biol. Chem. 2004; 279: 50329-50335Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 12Welling A. Ludwig A. Zimmer S. Klugbauer N. Flockerzi V. Hofmann F. Alternatively spliced IS6 segments of the α1C gene determine the tissue-specific dihydropyridine sensitivity of cardiac and vascular smooth muscle L-type Ca2+ channels.Circ. Res. 1997; 81: 526-532Crossref PubMed Scopus (192) Google Scholar) and can be altered under pathological conditions (13Yang Y. Chen X. Margulies K. Jeevanandam V. Pollack P. Bailey B.A. Houser S.R. L-type Ca2+ channel α 1c subunit isoform switching in failing human ventricular myocardium.J. Mol. Cell Cardiol. 2000; 32: 973-984Abstract Full Text PDF PubMed Scopus (71) Google Scholar, 14Liao P. Li G. Yu de J. Yong T.F. Wang J.J. Wang J. Soong T.W. Molecular alteration of Cav1.2 calcium channel in chronic myocardial infarction.Pflugers Arch. 2009; 458: 701-711Crossref PubMed Scopus (25) Google Scholar). In this report we screened the major alternatively spliced loci of Cav1.2 channels and identified a novel exon 33L in neonatal rat hearts that has not been reported previously. Inclusion of exon 33L generates a truncated channel Cav1.233L that does not conduct Ca2+. However, Cav1.233L produced a dominant-negative effect when co-expressed with functional Cav1.2 channels. Young adult male Wistar rats (2 months, 150–200 g) were sacrificed by CO2 and subsequent cervical dislocation. Neonatal hearts were harvested from postnatal day 1 rats of either sex. This study was approved and performed in accordance with the guidelines of the Institutional Animal Care and Use Committee of the University. Fetal human heart cDNA was purchased from Clontech. Adult human heart tissues were obtained from donors for heart transplantation in the National Heart Center of Singapore with the approval of the institutional review board. The work was approved by the Institutional Review Board committees of the National Heart Center of Singapore, Singapore General Hospital. Total RNA was isolated from the left ventricles. cDNA was generated from total RNA by incubating with reverse transcriptase and 18-mer oligo(dT). The PCR protocol includes: a denaturation step at 95 °C for 5 min; 35 cycles of 95 °C for 30 s, 52 °C or 55 °C for 45 s, and 72 °C for 1 min; a final extension step at 72 °C for 10 min. The primers used for the rat Cav1.2 exon 33L were: 5′-GCCTCTTCACGGTGGAG-3′ (forward) and 5′-TCCCAATCACTGCATAGATAA-3′ (reverse). The primers used for human Cav1.2 exon 33L are: 5′-CCAAGACCTAGAATACCGGG-3′ (forward) and 5′-CTACCACAGGGTGTTTCACC-3′ (reverse). The primers used for amplifying human Cav1.2 exons 18 to 24 as a control were: 5′-ATGAGGATAAGAGCCCCTACCC-3′ (forward) and 5′-ACTCGCAAGATCTTCACGACATTG-3′ (reverse). For single-cell RT-PCR, after electrophysiological characterization by the patch clamp method, the cells were collected into a vial containing reverse transcription reaction mix for first strand cDNA synthesis using the reverse primer. Single-cell PCR protocol was similar to RT-PCR, except for a 40-cycle amplification. This method was described previously to identify novel splice variations (7Tang Z.Z. Liang M.C. Lu S. Yu D. Yu C.Y. Yue D.T. Soong T.W. Transcript scanning reveals novel and extensive splice variations in human l-type voltage-gated calcium channel, Cav1.2 α1 subunit.J. Biol. Chem. 2004; 279: 44335-44343Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 14Liao P. Li G. Yu de J. Yong T.F. Wang J.J. Wang J. Soong T.W. Molecular alteration of Cav1.2 calcium channel in chronic myocardial infarction.Pflugers Arch. 2009; 458: 701-711Crossref PubMed Scopus (25) Google Scholar). In brief, the PCR products were cloned into pGEM-T Easy vector (Promega). After being transformed into DH10B Escherichia coli cells, each transformant was selected and grown in a single well in a 96-well plate. Colony PCR was performed with the same set of primers and conditions to identify the component of exons in each colony. Usually 96 colonies were selected for each sample. The genotype of each PCR product amplified from a colony was predicted by size and randomly confirmed by DNA sequencing. To differentiate mutually exclusive exons that are of same size, restriction enzyme digestions were used to identify the expression of individual exons. The digestion was carried out in a 30-μl reaction volume at 37 °C for 5 h. To control for complete digestion, the same amount of PCR product of a positive control DNA was subjected to similar digestion conditions. Rat exon 33L was verified by DNA sequencing. The sequences were compared with rat genome sequence from the Human Genome Sequencing Center, Baylor College of Medicine. To characterize the functions of exon 33L containing channels, a fragment containing exon 33L was cloned into a wild type rat Cav1.2 channel with AvrII and NheI sites. This wild type Cav1.2 channel contains the exons predominantly expressed in rat cardiac tissues. Rat exon 33L was cloned in-frame into pGEX-4T-1. Amino acid sequences from adjacent exons were not included. GST-fused protein was purified with glutathione-agarose (Sigma). Purified protein was used to immunize female New Zealand White rabbit once a month. Complete Freund's adjuvant was first mixed with GST-33L for immunization, and incomplete Freund's adjuvant was used in subsequent injections once a month. Serum collected after immunization was preabsorbed with GST protein first to remove GST antibodies, and 33L polyclonal antibody was affinity-purified from immobilized 33L protein with an IgG elution buffer (Pierce). The antibody concentration is 1 μg/μl. Serum from rabbit before immunization was used as preimmune control. For protein isolation, heart tissues were homogenized in HEPES lysis buffer: 20 mmol/liter HEPES, 137 mmol/liter NaCl, 1% Triton X-100, 10% glycerol, 1.5 mmol/liter MgCl2, and 1 mmol/liter EGTA supplemented with protease inhibitors (1:50 dilution, Roche Diagnostics). Homogenized samples were then centrifuged at 14,000 rpm for 15 min, and supernatants containing protein samples were collected. Protein concentration was determined using Bradford assay. 200 μg of lysates were separated by 8% SDS-PAGE and transferred overnight at 30 V onto PVDF membranes at 4 °C. Subsequently, the membrane was blocked with 1% BSA in 1× PBS + 0.1% Tween 20 for 1 h and probed with primary antibodies overnight at 4 °C. The primary antibodies used in the study include rabbit anti-exon 33L polyclonal (1:5000), rabbit anti-Cav1.2 or CaV1.3 (1:1000, Alomone), mouse anti-ubiquitin (1:1000, Invitrogen), and mouse anti-β-actin (1:5000, Sigma). The next day, the membrane was washed 3 times with 0.1% Tween 20 in 1× PBS. The membrane was then probed with HRP-conjugated secondary antibody for 1 h. After washing, the band was detected using Amersham Biosciences ECL Western blotting Analysis System (RPN2109, GE Healthcare). Animals were sacrificed and perfused with saline and subsequently 4% paraformaldehyde. The heats were then collected and post-fixed with 4% paraformaldehyde for 2 h. Dehydration was subsequently carried out by immersing the hearts into 15% sucrose followed by 30% sucrose. Next, the rat heart was sectioned at 20 μm of thickness. After washing with 0.2% Triton X-100 phosphate-buffered saline (PBST), 100 μl of blocking serum (10% goat serum and 1% bovine serum albumin in 0.2% PBST) was added onto the sections for 1 h. The sections were then incubated with primary antibodies overnight at 4 °C. Monoclonal antibodies anticonnexin 43 (MAB 3068) was purchased from Chemicon International. On the following day, tissue sections were washed 3 times with TNT wash buffer (0.1 m Tris-HCl buffer, pH 7.5, containing 0.15 m NaCl and 0.05% Tween 20). The slides were incubated with FITC-conjugated or Texas red-labeled secondary antibodies for 1 h at room temperature. After washing three times of wash buffer, the slides were mounted with FluorSaveTM reagent (Merck). The results were visualized using laser scanning confocal microscope system (Fluoview BX61, Olympus). The negative control was done in an identical procedure except for primary antibody incubation, and no positive signal was identified. HEK293 cells were transiently transfected with rat α1 (1.25 μg), β2a (1.25 μg), and α2δ (1.25 μg) subunits with the calcium phosphate transfection method. The GFP-tagged β2a and α2δ clones were provided by Dr. Terrance Snutch (University of British Columbia). The rat Cav3.2 channel and Cav1.3 channel were described earlier (15Tao J. Hildebrand M.E. Liao P. Liang M.C. Tan G. Li S. Snutch T.P. Soong T.W. Activation of corticotropin-releasing factor receptor 1 selectively inhibits CaV3.2 T-type calcium channels.Mol. Pharmacol. 2008; 73: 1596-1609Crossref PubMed Scopus (56) Google Scholar, 16Huang H. Yu D. Soong T.W. C-terminal alternative splicing of Cav1.3 channels distinctively modulates their dihydropyridine sensitivity.Mol. Pharmacol. 2013; 84: 643-653Crossref PubMed Scopus (35) Google Scholar). ICa was recorded with a whole-cell patch clamp technique at room temperature (∼25 °C) 48–72 h post-transfection. Patch electrodes were pulled using a Flaming/Brown micropipette puller (Sutter Instrument) and polished with a microforge. The external solution contained 140 mmol/liter tetraethylammonium methanesulfonate, 10 mmol/liter HEPES, and 1.8 mmol/liter CaCl2 (the pH was adjusted to 7.4 with CsOH and osmolarity to 300–310 mosm with glucose). For IBa recording, 1.8 mm Ca2+ was replaced by 5 mm Ba2+. The internal solution (pipette solution) contained 138 mmol/liter Cs-MeSO3, 5 mmol/liter CsCl2, 0.5 mmol/liter ethylene glycol tetraacetic acid, 10 mmol/liter HEPES, 1 mmol/liter MgCl2, and 2 mg/ml MgATP, pH 7.3 (adjusted with CsOH). The osmolarity was adjusted to between 290 and 300 mosm with glucose. Under voltage clamp and using an Axopatch 200B amplifier (Axon Instruments), whole-cell currents were filtered at 1–5 kHz and sampled at 5–50 kHz. The series resistance was normally <5 megaohms after compensation. The capacity transient was compensated using an online P/4 protocol. The steady-state inactivation curves were obtained from experiments by stepping from a holding potential of −90 mV to a 30-ms normalizing pulse to 10 mV followed by a family of 15-s-long prepulses from −80 to +20 mV. A 104-ms test pulse to +10 mV was recorded finally. Each test pulse was normalized to the maximal current amplitude of the normalizing pulse. The steady-state inactivation (SSI) 3The abbreviations used are: SSIsteady-state inactivationntnucleotide(s)NHneonatal heartAHadult heartpFpicofarad. data were fitted with a single Boltzmann equation, Irelative = Imin + (Imax − Imin)/(1 + exp((V½ − V)/k), where Irelative is the normalized tail current, V½ is the potential for half-inactivation, and k is the slope value. G-V curves were obtained from a tail activation protocol. The cells were activated by a 20-ms test pulse of variable voltage family from −60 to 100 mV, and tail currents were measured after repolarization to −50 mV for 10 ms. The tail currents were normalized to the peak currents before fitting with a dual Boltzmann equation. G/Gmax = Flow/{1 + exp((V½,low −V)/klow)} + (1 − Flow)/{1 + exp((V½,high − V)/khigh)}, where G is the tail current, and Gmax is the peak tail current, Flow is the fraction of low threshold component, and V½,low, V½,high, klow, and khigh are the half-activation potentials and slope factors for the low and high threshold components, and V½act was calculated when G = 0.5Gmax. steady-state inactivation nucleotide(s) neonatal heart adult heart picofarad. Cav1.2 channel undergoes extensive alternative splicing, which affects its tissue distribution, pharmacology, and electrophysiological properties (4Liao P. Yong T.F. Liang M.C. Yue D.T. Soong T.W. Splicing for alternative structures of Cav1.2 Ca2+ channels in cardiac and smooth muscles.Cardiovasc. Res. 2005; 68: 197-203Crossref PubMed Scopus (102) Google Scholar, 10Abernethy D.R. Soldatov N.M. Structure-functional diversity of human L-type Ca2+ channel: perspectives for new pharmacological targets.J. Pharmacol. Exp. Ther. 2002; 300: 724-728Crossref PubMed Scopus (80) Google Scholar). Although up to 20 exons are alternatively spliced (17Liao P. Zhang H.Y. Soong T.W. Alternative splicing of voltage-gated calcium channels: from molecular biology to disease.Pflugers Arch. 2009; 458: 481-487Crossref PubMed Scopus (37) Google Scholar), only exons 31 and 32 have been reported to be developmentally regulated in the heart (18Diebold R.J. Koch W.J. Ellinor P.T. Wang J.J. Muthuchamy M. Wieczorek D.F. Schwartz A. Mutually exclusive exon splicing of the cardiac calcium channel α1 subunit gene generates developmentally regulated isoforms in the rat heart.Proc. Natl. Acad. Sci. U.S.A. 1992; 89: 1497-1501Crossref PubMed Scopus (91) Google Scholar). Mutually exclusive exons 31 and 32 encode the IVS3 transmembrane segment and part of the IVS2-S3 intracellular linker, whereas another alternatively spliced exon 33 encodes the IVS3-S4 extracellular linker. There exist extensive alternative splicings within this region (7Tang Z.Z. Liang M.C. Lu S. Yu D. Yu C.Y. Yue D.T. Soong T.W. Transcript scanning reveals novel and extensive splice variations in human l-type voltage-gated calcium channel, Cav1.2 α1 subunit.J. Biol. Chem. 2004; 279: 44335-44343Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar) involved in many cardiovascular diseases (6Tiwari S. Zhang Y. Heller J. Abernethy D.R. Soldatov N.M. Atherosclerosis-related molecular alteration of the human Cav1.2 calcium channel α1C subunit.Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 17024-17029Crossref PubMed Scopus (72) Google Scholar, 13Yang Y. Chen X. Margulies K. Jeevanandam V. Pollack P. Bailey B.A. Houser S.R. L-type Ca2+ channel α 1c subunit isoform switching in failing human ventricular myocardium.J. Mol. Cell Cardiol. 2000; 32: 973-984Abstract Full Text PDF PubMed Scopus (71) Google Scholar, 14Liao P. Li G. Yu de J. Yong T.F. Wang J.J. Wang J. Soong T.W. Molecular alteration of Cav1.2 calcium channel in chronic myocardial infarction.Pflugers Arch. 2009; 458: 701-711Crossref PubMed Scopus (25) Google Scholar, 19Gidh-Jain M. Huang B. Jain P. Battula V. el-Sherif N. Reemergence of the fetal pattern of L-type calcium channel gene expression in non infarcted myocardium during left ventricular remodeling.Biochem. Biophys. Res. Commun. 1995; 216: 892-897Crossref PubMed Scopus (59) Google Scholar). RT-PCR across exons 30–35 will generate a 382-bp amplicon containing exon 33 and one of the mutually exclusive exons 31 or 32. Enzyme digestion on exon 32 by NsiI produces two smaller fragments with the sizes of 68 and 314 bp, respectively (Fig. 1A). In the presence of exon 31, only one band exists (382 bp), as no digestion occurs. Deletion of the cassette exon 33 (33 nt in length) has rarely been found in rat hearts (8Liao P. Yu D. Li G. Yong T.F. Soon J.L. Chua Y.L. Soong T.W. A smooth muscle Cav1.2 calcium channel splice variant underlies hyperpolarized window current and enhanced state-dependent inhibition by nifedipine.J. Biol. Chem. 2007; 282: 35133-35142Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). When we digested RT-PCR products from the neonatal and adult rat hearts, partial digestions were observed in both samples, indicating that neonatal and adult hearts contain a mixture of exons 31 and 32. However, the extent of digestion by NsiI was different, as more PCR products from the adult hearts were digested than that from the neonatal hearts. Therefore, exon 32 expression is higher in adult rat hearts than in neonatal rat hearts (Fig. 1B). Such developmental changes of exon 31 and 32 have been reported by Diebold et al. (18Diebold R.J. Koch W.J. Ellinor P.T. Wang J.J. Muthuchamy M. Wieczorek D.F. Schwartz A. Mutually exclusive exon splicing of the cardiac calcium channel α1 subunit gene generates developmentally regulated isoforms in the rat heart.Proc. Natl. Acad. Sci. U.S.A. 1992; 89: 1497-1501Crossref PubMed Scopus (91) Google Scholar), and our experiment supports the previous finding. Besides mutually exclusive exons 31, 32, and cassette exon 33, there exists an additional 66-nt extension at the 5′ end of exon 34 with different acceptor sites, producing exon 34a, an isoform of exon 34 (6Tiwari S. Zhang Y. Heller J. Abernethy D.R. Soldatov N.M. Atherosclerosis-related molecular alteration of the human Cav1.2 calcium channel α1C subunit.Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 17024-17029Crossref PubMed Scopus (72) Google Scholar). To characterize the detailed expression of exon 33 and 34a, we used the colony screening method that is able to detect exons of lower expression (8Liao P. Yu D. Li G. Yong T.F. Soon J.L. Chua Y.L. Soong T.W. A smooth muscle Cav1.2 calcium channel splice variant underlies hyperpolarized window current and enhanced state-dependent inhibition by nifedipine.J. Biol. Chem. 2007; 282: 35133-35142Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). The inclusion or exclusion of exons 33 and/or 34a could be easily identified by differences in size of the PCR products, whereas mutually exclusive exons 31 and 32 cannot, as they are of the same length. Colony PCR screening showed that the size of the majority of colonies was ∼382 bp, indicating that the splice variants with exon 33 deletion or exon 34a inclusion are not predominant in neonatal and adult rat hearts (Fig. 2, A and B). DNA sequencing confirmed that the colonies with smaller size contained only exon 31 or 32 but with the exon 33 deletion. However, some colonies of a larger size have been found to appear more frequently in the neonatal heart than in the adult heart (indicated by arrows in Fig. 2, A and B). Subsequent DNA sequencing confirmed that these colonies did not contain exon 34a, but expressed a novel exon, which is named exon 33L here. We repeated RT-PCR-based colony screening experiments and confirmed that neonatal hearts (NH) express 9.7% of exon 33L, which represents a >2-fold increase to the 4.3% in adult hearts (AH) (Fig. 2C, p = 0.0127, NH (n = 5), AH (n = 5), Student's t test). Exon 33L contains an additional 71-nt 5′ to exon 33 (Fig. 3A). To understand the nature of this 71-nt fragment, we searched the rat genome sequence from the Human Genome Sequencing Center from Baylor College of Medicine. The mechanism for the generation of the additional 71-nt 5′ of exon 33 can be rationalized by the inspection of the genomic sequence of the rat Cav1.2 channel gene. These 71 nt locate at chromosome 4 and 5′ to exon 33 of the rat Cav1.2 gene. No genomic sequence was found between the 71 nt and exon 33. More importantly, the canonical -ag- acceptor dinucleotides were found 5′ to the 71 nt. Therefore, the inclusion of the 71 nt could be explained by the use of an alternate acceptor site of exon 33. However, the inclusion of 71 nt will result in a frameshift of the coding sequences and produce a premature stop codon (Fig. 3, A and B). As the premature stop codon appeared in the IVS3-4 linker, it will generate a truncated channel lacking the last three transmembrane segments of domain IV and the entire C terminus (Fig. 3C). Thus, there exists two isoforms of exon 33. One is the original 33-nt exon 33 and the other is the new 104-nt exon 33L (71 nt + 33 nt). We further compared the DNA sequencing results and found that exon 33L can be linked either with exon 31 or exon 32. To determine whether the aberrant splice variants are translated into proteins, a polyclonal antibody was generated against the 22-amino acid polypeptides encoded by exon 33L (Fig. 3A). The 22-amino acid polypeptides were expressed as a fusion protein with GST (Fig. 4A) and were injected into rabbits to produce exon 33L specific antibody α-33L. No adjacent amino acids were included for antibody production. Using α-33L, we detected by Western blot a band of 160 kDa in neonatal and adult hearts, having the same molecular mass of the truncated 33L containing Cav1.2 channel expressed in HEK 293 cells (Fig. 4B). Therefore, neonatal rat hearts expressed higher levels of Cav1.233L channel than adult hearts. To prove the specificity of α-33L antibody, HEK cells were transfected with Cav1.233L channel or the reference channel Cav1.2WT, which does not contain exon 33L. α-33L antibody stained Cav1.233-transfected cells but not Cav1.2WT-transfected cells (Fig. 4C). Next, we used an immunohistological method to examine the cellular expression of the Cav1.233L channel in rat hearts during development. Connexin 43 was labeled for the gap junctions between adjacent cardiac muscle cells. Again, α-33L staining showed a stronger fluorescent signal in neonatal heart than in adult heart, supporting a higher expression of Cav1.233L channel in neonatal rat heart (Fig. 4D). It should be noted that Cav1.233L did not colocalize with Connexin 43 in both neonatal and adult hearts, suggesting that the majority of Cav1.233L channels may not traffic to the plasma membrane. The functional significance of the inclusion of exon 33L in Cav1.2 channels has not been reported. We sought to assess exon 33L functions by whole-cell patch clamp recording. Exon 33L was cloned into a reference rat cardiac-form Cav1.2 channel that contained exon 1a and 8a, with the absence of exon 9* (8Liao P. Yu D. Li G. Yong T.F. Soon J.L. Chua Y.L. Soong T.W. A smooth muscle Cav1.2 calcium channel splice variant underlies hyperpolarized window curr" @default.
• W2017492952 created "2016-06-24" @default.
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• W2017492952 date "2015-04-01" @default.
• W2017492952 modified "2023-09-30" @default.
• W2017492952 title "Alternative Splicing Generates a Novel Truncated Cav1.2 Channel in Neonatal Rat Heart" @default.
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