Ca channel-dependent Ca flux plays a critical role in control of cardiac excitation-contraction coupling. However, we have limited understanding of how the abundance of functional Ca channels in the sarcolemmal is regulated. The goal of these studies is to examine mechanisms by which innervation and transmembrane signalling regulates expression and function of cardiac Ca channels. We will seek intracellular effectors that control the abundance of the Ca channel protein, dihydropyridine binding sites, and functional Ca channels. The principal experimental model will be cultured chick myocytes. Initially, we will purify partially the cardiac Ca channel and develop antibodies to it. Monoclonal antibodies will be used to examine the distribution of Ca channels and the relation between dihydropyridine (DHP) binding sites and channel protein expression. Ca channel function will be studied with a fluorescence microscopy-video motion analysis system and related to abundance of immunoreactive Ca channels and DHP binding sites. Using these pharmacological and functional probes, the hypothesis will be tested that expression of beta-adrenergic receptors and Ca channels can be coregulated. We will test the related hypothesis that expression of Ca channel protein can be specifically regulated by a protein kinase A-dependent mechanism. It will be determined whether receptor occupancy is necessary and whether activation of protein kinase A is sufficient to alter Ca channel expression. It will then be determined whether subsequent alterations in (Ca)i is necessary for channel regulation. The hypotheses will then be tested that cardiac sympathetic innervation regulates Ca channel expression by tonically altering (cAMP) or (Ca)i, or whether other trophic factors regulate the Ca channel. Finally, we will determine whether extracellular stimuli that activate protein kinase C regulate the abundance of immunoreactive and functional Ca channels. Common regulatory pathways for protein kinase C and protein kinase A will be sought. Ca channels are widely distributed among excitable and secretory cells. New insights into fundamental mechanisms of Ca channel regulation gained from these investigations will inform our approaches to therapy for cardiovascular disease, as well as disease of other systems.
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