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

Abstract

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].

Unchanged β-Adrenergic Stimulation of Cardiac L-type Calcium Channels in Cav1.2 Phosphorylation Site S1928A Mutant Mice

“…Taken together, our results support the notion that PKA phosphorylation of Ser 1928 of Ca v 1.2 is functionally not involved in ␤-adrenergic regulation of I CaL in murine ventricular cardiomyocytes. The results are in line with the previous finding that ␤-adrenergic stimulation requires a PKAmediated phosphorylation step (1,6,9,11,13,16,18,24). Many questions remain to understand the ␤-adrenergic regulation of Ca 2ϩ channels.…”

“…In contrast to these reports, Ganesan et al (18) postulated that at least 70% of the ␤-adrenergic regulation of I CaL in virally transducted heart cells cannot be attributed to the Ser 1928 phosphorylation event. However, the viral infection system by Ganesan et al (18) only partially reconstituted the regulation of I CaL (the PKA-mediated increase in current was only 50%).…”

“…However, the viral infection system by Ganesan et al (18) only partially reconstituted the regulation of I CaL (the PKA-mediated increase in current was only 50%).…”

β-Adrenergic Stimulation of L-type Ca 2+ Channels in Cardiac Myocytes Requires the Distal Carboxyl Terminus of α 1C but Not Serine 1928

“…23 A serine to alanine substitution of ␣ 1C(D-) at S1928 (␣ 1C(D-)-S1928A ) was constructed by site-directed mutagenesis with overlapping primers, using the QuikChange XL kit (Stratagene). A carboxyl-terminal truncation mutant, pAdEcd-␣ 1C(D-)⌬1905 , in which the carboxyl terminus of the ␣ 1C was truncated after amino acid 1905, was also created.…”

The PDZ Motif of the α1C Subunit Is Not Required for Surface Trafficking and Adrenergic Modulation of CaV1.2 Channel in the Heart

“…Cultured neurons transfected, using a Ca 2ϩ phosphate technique, with ␣ 1C lacking the PDZ ligand domain exhibited attenuated cAMP-response element-binding protein responses, indicating an important role for the PDZ ligand motif in the subcellular localization of ␣ 1C in neurons (21). Although adenoviruses have been used to express Ca V 1.2 subunits in cardiomyocytes (35,36), creation of adenoviruses encoding ␣ 1C is difficult because of the ␣ 1C insert size and because viral infection requires that cardiomyocytes be cultured for extended periods. Although useful for investigating biophysical properties, many relevant aspects of Ca V 1.2 channel targeting and functional modulation in heart cannot be studied in heterologous expression systems, which lack the complex cytoarchitecture and intracellular environment of adult cardiomyocytes.…”