Considerable progress on skeletal muscle L-type Ca2+ channels has been made at the pharmacological, biophysical, biochemical, and molecular biological levels. In fact, all the known subunits of the skeletal muscle L-type Ca2+ channel have been purified and cloned (alpha1, alpha2, beta, gamma, delta). However, less is known of the subunit structure of the cardiac channel. I will focus my research on the cardiac Ca2+ channel, since this channel plays a pivotal role in both the physiology and pathophysiology of the cardiovascular system, as well as being the site of action of a number of clinically relevant drugs, the """"""""calcium antagonists."""""""" I have discovered that the gene encoding the a, subunit of the cardiac Ca2+ channel is alternatively spliced to produce at least three transcripts. I hypothesize that these transcripts encode L-type Ca2+ channel subtypes that differ in their biophysical properties.
The first aim of this project is to characterize the electrophysiological properties of these alpha1 subtypes using the Xenopus laevis oocyte expression system. The second hypothesis I will test is that the other Ca2+ channel subunits modulate the biophysical properties of the alpha1 subunit. I plan to clone the other subunits of the cardiac L-type Ca2+ channel using the skeletal muscle cDNA's as probe, then coexpress them with alpha1. Since the hormonal regulation of cardiac contractility is in part mediated by phosphorylation of the L-type Ca2+ channel, my third aim is to study how these channels are regulated by phosphorylation. Since the submission of my original grant application I have achieved considerable progress on these research goals. I have optimized and partially characterized the expression of a cardiac alpha1 subtype in oocytes using the 2 microelectrode voltage clamp. In addition, I have cloned a new beta subunit that is expressed in heart and brain, and shown that it can modulate the biophysical properties of alpha1. These preliminary studies demonstrate the feasibility of the proposed studies, as well as my ability to carry them out.
