| The alpha1C subunit of the cardiac L-type Ca2+ channel, which contains the channel pore, voltage- and Ca2+-dependent gating structures, and drug-binding sites, has been well-studied in heterologous expression systems. However, many aspects of L-type Ca 2+ channel behavior in intact cardiomyocytes remain incompletely characterized, including the physiological sites of phosphorylation and the influence of various structural motifs on cardiac excitation-contraction coupling. In this dissertation, the development of adenoviral constructs with E1, E3 and fiber gene deletions is described. The additional fiber gene deletion was designed to allow incorporation of full-length alpha1C gene cassettes into the adenovirus backbone. Wild type (alpha1C-WT) and dihydropyridine (DHP) insensitive-(alpha1C-D-) Ca2+ channel adenoviruses were constructed. These alpha1C adenoviruses were expressed in A549 cells and adult-guinea pig cardiac myocytes, and L-type Ca2+ currents (ICaL) measured by whole-cell voltage clamp. ICaL carried by alpha1C-D- was markedly less sensitive to nitrendipine than alpha1C-WT, a feature exploited to discriminate between engineered and native currents in transduced guinea-pig myocytes.; In the second part of this dissertation, DHP-insensitive alpha 1C adenoviruses were used to study the molecular mechanism of beta-adrenergic receptor regulation of cardiac L-type Ca2+ channels. Previous reports have shown in vitro phosphorylation of a consensus protein kinase A (PKA) site at serine 1928 on the carboxyl-terminus of the alpha 1C subunit. However, the functional role of this site has not been investigated in cardiac myocytes. The effects of truncating the distal carboxyl-terminus of the alpha1C subunit at amino acid residue 1905 or mutating the putative PKA site at serine 1928 to alanine, were examined in adult myocytes, using DHP-insensitive alpha1C adenoviruses. Expression of alpha 1C truncated at 1905 dramatically attenuated the increase of peak I CaL induced by isoproterenol. However, the point mutation S1928A did not significantly affect the beta-adrenergic response. The findings indicate that the distal carboxyl-terminus of alpha1C plays an important role in beta-adrenergic upregulation of cardiac L-type Ca2+ channels, but that phosphorylation of serine 1928 is not required for this effect. |
