Regulatory Mechanisms of Cardiac Repolarization

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DESCRIPTION (provided by applicant): Voltage-gated K+ channels (Kv channels) play an important role in setting the resting potential and determining repolarization in the cardiovascular system. Recent evidence suggests that specialized microdomains commonly referred to as lipid rafts exist within the plane of most plasma membranes. These domains are enriched in cholesterol and sphingolipids and concentrate a number of signal transduction molecules. In the Preliminary Data section, we demonstrate that the voltage-gated K+ channels, Kv2.1, Kv1.1, Kv1.5, and Kv1.4, but not Kv4.2, target to lipid rafts in both heterologous expression systems and the rat cardiovascular system and brain. In addition, Kv2.1 and Kv1.5 reside in different raft compartments, with Kv1.5 specially targeting to caveolae. Demonstrating that cardiac rafts contain proteins other than voltage-gated ion channels, we have also localized connexin-43 to lipid rafts in both adult heart and cultured neonatal myocytes. Depletion of cellular cholesterol alters the buoyancy of the Kv2.1-associated rafts and shifts the midpoint of Kv2.1 inactivation by nearly 40 mV without affecting peak current density or channel activation. In addition, the preliminary data suggest channel targeting and cell surface localization involve the association with specific raft compartments. Thus, raft association is significant from both a functional and localization standpoint. This application focuses on the link between both Kv channels and connexins in the cardiovascular system. The Specific Aims will 1) characterize Kv channel association with lipid rafts in cardiac and vascular tissue, 2) address the mechanisms involved in the targeting of Kv1.5 to caveolae, with emphasis placed on alpha subunit composition and transmembrane domains, 3) examine the functional significance of Kvchannel association with lipid raft domains, 4) determine the relationship between raft integrity and gap junction function and localization in cultured myocytes, and 5) isolate the individual cardiac intercalated disk regions containing Kv1.5 and connexins in order to identify the associated proteins important to channel function and localization. Given the role that both Kv channels and connexins play in cardiac and vascular smooth muscle, the proposed research will significantly advance our understanding of electrical excitability in the cardiovascular system.