Evidence for the existence of a cardiac specific isoform of the α<sub>1</sub> subunit of the voltage dependent calcium channel

Slish, Donald F.; Engle, Dorothy; Váradi, Gyula; Lotan, Ilana; Singer, Datna; Dascal, Nathan; Schwartz, Arnold

doi:10.1016/0014-5793(89)80786-0

Cited by 60 publications

(24 citation statements)

References 25 publications

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“…The full-length rabbit α 1c subunit (with the alternatively spliced domain in IVS3, as previously described [12]) was ligated into pAdEcd, an adenovirus shuttle vector containing an ecdysone inducible promoter [13], to make pAdEcd-α 1c-wt .…”

Section: Experimental Procedures α 1c-d-plasmid Preparationmentioning

confidence: 99%

Reverse engineering the L-type Ca2+ channel α1c subunit in adult cardiac myocytes using novel adenoviral vectors

Ganesan

O’Rourke

Maack

et al. 2005

Biochemical and Biophysical Research Communications

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The α 1c subunit of the cardiac L-type Ca 2+ channel, which contains the channel pore, voltage-and Ca 2+ -dependent gating structures, and drug binding sites, has been well studied in heterologous expression systems, but many aspects of L-type Ca 2+ channel behavior in intact cardiomyocytes remain poorly characterized. Here, we develop adenoviral constructs with E1, E3 and fiber gene deletions, to allow incorporation of full-length α 1c gene cassettes into the adenovirus backbone. Wildtype (α 1c-wt ) and mutant (α 1c-D-) Ca 2+ channel adenoviruses were constructed. The α 1c-D-contained four point substitutions at amino acid residues known to be critical for dihydropyridine binding. Both α 1c-wt and α 1c-D-expressed robustly in A549 cells (peak L-type Ca 2+ current (I CaL ) at 0 mV: α 1c-wt −9.94 ± 1.00 pA/pF, n = 9; α 1c-D-−10.30 pA/pF, n = 12). I CaL carried by α 1c-D-was markedly less sensitive to nitrendipine (IC 50 17.1 µM) than α 1c-wt (IC 50 88 nM); a feature exploited to discriminate between engineered and native currents in transduced guinea-pig myocytes. 10 µM nitrendipine blocked only 51 ± 5% (n = 9) of I CaL in α 1c-D--expressing myocytes, in comparison to 86 ± 8% (n = 9) of I CaL in control myocytes. Moreover, in 20 µM nitrendipine, calcium transients could still be evoked in α 1c-D--transduced cells, but were largely blocked in control myocytes, indicating that the engineered channels were coupled to sarcoplasmic reticular Ca 2+ release. These α 1c adenoviruses provide an unprecedented tool for structure-function studies of cardiac excitationcontraction coupling and L-type Ca 2+ channel regulation in the native myocyte background. KeywordsDihydropyridine; Excitation-contraction coupling; Gene transfer; Adenovirus; Ion channelThe cardiac L-type Ca 2+ channel (Ca v 1.2a) is a multisubunit complex of five proteins located on the surface membrane of the cardiac myocyte. The largest component is the pore-forming α 1 subunit (∼220 kDa), the critical determinant of most functional properties of the channel, including voltage-dependent gating, Ca 2+ permeability, Ca 2+ -dependent inactivation [1,2], and inhibition by the three principal classes of Ca 2+ channel antagonists [3][4][5].

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Section: Experimental Procedures α 1c-d-plasmid Preparationmentioning

confidence: 99%

Reverse engineering the L-type Ca2+ channel α1c subunit in adult cardiac myocytes using novel adenoviral vectors

Ganesan

O’Rourke

Maack

et al. 2005

Biochemical and Biophysical Research Communications

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“…␣ 1C Subunits with Rabbit Heart-type N Terminus Should Be Widespread-Alternative splicing products of ␣ 1C are found in various tissues; they have been proposed to play an important role in generating a diversity of electrophysiological properties (14,42,62,73,75). Alternative splicing was also proposed to take place in the N terminus, and "rabbit heart," "rat brain," and "lung" cDNAs have been assumed to be splice variants of the same gene product (42,62).…”

Section: N Terminus In Cardiac Ca2ϩ Channel Function and Modulationmentioning

confidence: 99%

Crucial Role of N Terminus in Function of Cardiac L-type Ca2+ Channel and Its Modulation by Protein Kinase C

Shistik

Ivanina

Blumenstein

et al. 1998

Journal of Biological Chemistry

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101

124

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In the heart, Ca 2ϩ current via the voltage-dependent L-type channels (dihydropyridine-sensitive) underlies the plateau of the action potential and provides calcium ions necessary for initiation of cardiac cell contraction (2). Similar channels are found in smooth muscle, where they play a major role in regulation of tonus and contraction (3, 4), and in the nervous system (5, 6). L-type channels are composed of the following three subunits: the main, pore-forming ␣ 1C , the cytosolic ␤2, and the ␣ 2 ␦ subunit which is mostly extracellular (5, 7-11). ␣ 1C contains four homologous membrane domains numbered I-IV, each one with six transmembrane segments and a re-entrant P-loop that forms the pore lining; N-and C-terminal domains and the linkers connecting the domains I-II, II-II, and II-IV are cytosolic (see Ref. 7 for review, and see Fig. 6A for a scheme). The C terminus was implicated in Ca 2ϩ -and voltagedependent inactivation (12-15) and modulation by protein kinase A (16 -19); linker I-II contains the binding site for the ␤ subunit (20, 21).Cardiac and smooth muscle L-type channels are tightly regulated by hormonal and neuronal signals via G proteins and protein kinases (22,23). Protein kinase C (PKC) 1 is one of such regulators; its actions appear to be tissue-and species-specific. PKC activators, such as phorbol esters and diacylglycerols, increase Ca 2ϩ channel currents in cardiac and smooth muscle cells of various mammals (24 -33), and PKC has been implicated in mediating the stimulation of Ca 2ϩ channels by intracellular ATP (34), angiotensin II (26), glucocorticoids (28), pituitary adenylate cyclase-activating polypeptide (33), and arginine-vasopressin (32). PKC up-regulation results from changes in channel gating because it is accompanied by an increase in single channel open probability, P o (30,35,36). In many cases, a biphasic effect of PKC activators has been described, with an increase followed by a later decrease (25,27,30), and some preparations such as adult guinea pig heart cells (37, 38) respond to phorbol esters only by a decrease in Ca 2ϩ currents, an effect that may not be mediated by PKC (38). The biphasic response to PKC stimulators is fully reconstituted when expression of L-type channels in Xenopus oocytes is directed by RNA extracted from rat heart (39, 40) or cRNA of rabbit cardiac ␣ 1C subunit (39). Increase of Ca 2ϩ channel activity by phorbol esters has also been observed in a mammalian cell line (baby hamster kidney) expressing the rabbit cardiac ␣ 1C (36). The potentiation by phorbol esters of Ca 2ϩ channels expressed in the oocytes is mediated by PKC because it is mimicked by diacylglycerols and blocked by specific PKC inhibitors (39,40).Both ␣ 1C and ␤ are substrates for PKC-catalyzed phosphorylation (Ref. 41 and references therein). ␣ 1C subunit has been recognized as the target for the Ca 2ϩ channel enhancement caused by PKC, since coexpression of the auxiliary subunits was not necessary to reproduce the effect of phorbol esters; on the contrary, coexpression of the ␤ subunit...

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“…Clones isolated from rabbit heart and lung (8,12,16), rat brain (19), and human heart (20) also display the same sequence variability within this particular S3 region. Interestingly, the variable IVS3 region in the rat brain is generated from a gene distinct from the one expressed in rat heart and smooth muscle (19).…”

Section: Introductionmentioning

confidence: 99%

Mutually exclusive exon splicing of the cardiac calcium channel alpha 1 subunit gene generates developmentally regulated isoforms in the rat heart.

Diebold

Koch

Ellinor

et al. 1992

Proc. Natl. Acad. Sci. U.S.A.

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Several clones were isolated from a rat genonic library in order to further characterize a region of variability within the third membrane-spanning region of the fourth motif (IVS3) of the L-type voltage-dependent calcium channel. We report here that this diversity arises from alternative splicing of a primary transcript containing a single pair of adjacent exons each encoding a unique sequence for the IVS3 region. Definitive proof of a mutually exclusive splicing mechanism was obtained by genomic mapping of flanking upstream and downstream exons and by extensive sequence analysis of the relevant exon/intron boundaries. S1 nuclease protection experiments revealed that both variant forms of the IVS3 were equally expressed in newborn and fetal rat heart, whereas only a single isoform predominated in adult rat heart. The results demonstrate the existence of an important developmentally regulated switch mediated by alternatively spliced exons in cardiac tissue at a time when major changes in excitation occur.Voltage-dependent calcium channels (VDCCs) are a diverse class of molecules found in all excitable cells (1-4). These channels are essential for many cellular functions such as muscle contraction, propagation of action potentials, maintenance of electrical activity, excitation-secretion coupling, and neurotransmitter regulation (2, 3). The L-type VDCC from rabbit skeletal muscle has been purified and extensively characterized biochemically. The channel exists as a pentameric macromolecule consisting of a1, a<-8, 3,, and y subunits, all of which have been cloned and sequenced (4-7).Functionally, the a1 subunit alone can act as a VDCC (8, 9) and contains binding sites for calcium antagonists (10, 11), although native gating characteristics are lacking (9). The a2-8 and, in some cases, P subunits significantly increase current amplitude displayed by expressed a1 subunits (8,(12)(13)(14). More importantly, the 83 subunit modulates gating properties of the expressed skeletal muscle a1 subunit in mammalian L-cells (14). In Xenopus oocytes, the expression of the skeletal muscle 'y subunit determines the inactivation properties of the coexpressed cardiac a1 subunit (15). Thus, a normal functioning calcium channel probably requires the presence of all the other subunits.The a1 subunits from rabbit cardiac muscle and lung (8, 13, 16), rat aorta (17), and carp skeletal muscle (18) have been cloned and their primary structures have been described.Like the skeletal muscle a1 subunit, the cardiac and smooth muscle isoforms are large hydrophobic polypeptides comprising four repeating motifs (I-IV), each containing six putative transmembrane segments (S1-S6), flanked by Nand C-terminal cytoplasmic domains. Sequence analysis suggests that the cardiac and smooth muscle a1 subunits are encoded by the same gene, which is distinct from that encoding the skeletal muscle channel (9,12,17).In the cloning of the rat aorta a1 subunit, we described the existence of two cDNA species that were identical in all but the third membrane...

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Evidence for the existence of a cardiac specific isoform of the α₁ subunit of the voltage dependent calcium channel

Cited by 60 publications

References 25 publications

Reverse engineering the L-type Ca2+ channel α1c subunit in adult cardiac myocytes using novel adenoviral vectors

Reverse engineering the L-type Ca2+ channel α1c subunit in adult cardiac myocytes using novel adenoviral vectors

Crucial Role of N Terminus in Function of Cardiac L-type Ca2+ Channel and Its Modulation by Protein Kinase C

Mutually exclusive exon splicing of the cardiac calcium channel alpha 1 subunit gene generates developmentally regulated isoforms in the rat heart.

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Evidence for the existence of a cardiac specific isoform of the α1 subunit of the voltage dependent calcium channel

Cited by 60 publications

References 25 publications

Reverse engineering the L-type Ca2+ channel α1c subunit in adult cardiac myocytes using novel adenoviral vectors

Reverse engineering the L-type Ca2+ channel α1c subunit in adult cardiac myocytes using novel adenoviral vectors

Crucial Role of N Terminus in Function of Cardiac L-type Ca2+ Channel and Its Modulation by Protein Kinase C

Mutually exclusive exon splicing of the cardiac calcium channel alpha 1 subunit gene generates developmentally regulated isoforms in the rat heart.

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Evidence for the existence of a cardiac specific isoform of the α₁ subunit of the voltage dependent calcium channel