Ion channel regulation by macromolecular complexes

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DESCRIPTION (provided by applicant): Modulation of ion channels by macromolecular signaling complexes and phosphorylation plays an important role in the regulation of excitation-contraction (E-C) coupling. In cardiac myocytes, the L-type Ca2+ channel (Cav1.2) current (ICa), the major depolarizing current contributing to the plateau of the action potential and E-C coupling, is highly regulated by hormones, in large part through the activation of protein kinases and phosphatases. It is well established that Cav1.2 plays a key role in modulating cardiac function in response to classical signaling pathways, such as the renin-angiotensin system (RAS) and sympathetic nervous system (SNS). Treatment of several major cardiovascular diseases is dependent, in part, upon the modulation of these pathways by drugs. This proposal focuses on gaining a better understanding of the mechanisms by which a major cardiac protein kinase, protein kinase C (PKC), which is activated by the RAS and SNS, modulates the function of Cav1.2. We have mapped critical PKC phosphorylation sites on the Cav1.2 alpha1c and beta2 subunits and have prepared phospho-peptide specific antibodies; each uniquely designed to recognize only a single phosphorylated Cav1.2 channel site. Our preliminary data suggest that the different cardiac PKC isoforms can phosphorylate distinct Cav1.2 sites in vitro. Utilizing the phospho-peptide and site specific antibodies, the applicant proposes to study the role of different PKC isoforms in the modulation of Cav1.2 in normal and pathological hearts. Three specific aims are proposed: (1) To characterize biochemically and pharmacologically the phosphorylation of Cav1.2 in cells, tissues and animals; (2) To characterize electrophysiologically PKC modulation of cardiac Cav1.2; (3) To characterize the PKC modulation of Cav1.2 in heart failure and hypertrophy. Utilizing biochemical, molecular biological, and electrophysiological techniques, we will explore the molecular mechanisms and cellular signaling pathways impinging upon Cav1.2 regulation in normal and diseased hearts. Electrophysiological characterization of the effects of PKC phosphorylation of specific Cav1.2 residues will be carried out in heterologous expression systems and cardiomyocytes. Since heart failure and hypertrophy are associated with alterations in PKC activity and Ca2+ homeostasis, understanding the PKC modulation of Cav1.2 may contribute to the development of novel therapeutic agents to treat these disorders.