The long-term goal of the proposed research is to understand the mechanisms by which ion channels are targeted to specific membrane microdomains in cells of the cardiovascular system and the functional significance of this precise subcellular organization. The electrical and contractile properties of both the heart and vascular smooth muscle are dependent on the expression of voltage-gated potassium (Kv) channels that regulate the cardiac action potential and control arterial tone. Most tissues, and even single cells within the cardiovascular system, express multiple Kv channel types belonging to one or more subfamilies. Importantly, the subcellular localization of these different channel isoforms is critical for proper cell function and signaling. Progress has been made in identifying elements involved in channel targeting, clustering and anchoring. However, it is not yet clear how the number and location of channel complexes within the plane of the membrane are determined or how this compartmentalization affects channel function. Historically, K+ channel targeting and localization was thought to involve primarily protein-protein interactions between channels and PDZ-domain-containing scaffolding proteins or the actin cytoskeleton. Recently however, we showed for the first time that Kv channels target to specialized lipid microdomains within the plane of the plasma membrane. We propose that interactions between channel protein and these "lipid raft" microdomains represent a novel mechanism of channel targeting and for modulating channel properties via alterations in lipid content. We have developed a unique model system in which it is possible to demonstrate isoform-specific localization of Kv channels to distinct lipid raft populations, with the novel finding that Kv1.5 localized to caveolae. This system offers a unique opportunity to study basic mechanisms of channel targeting in vitro. Preliminary results in vivo indicate that Kv1.5 is found in the raft fractions in both rat heart and aorta. In addition, Western blot analysis indicates that post/cotranslational modifications of Kv1.5 may target channel protein to caveolar microdomains. Furthermore, preliminary experiments indicate that depletion of raft lipid alters Kv1.5 channel function and that these effects may involve a disruption of channel/tyrosine kinase interaction. Based on these data, we propose to investigate the following Specific Aims: 1) Establish the association of Kv1.5 with lipid raft microdomains in vascular smooth muscle; 2) Determine the mechanisms by which Kv1.5 is targeted to lipid rafts and caveolae; 3) Demonstrate that the localization of Kv1.5 to caveolae is necessary for proper channel function and/or regulation. The proposed research will significantly advance our understanding of the regulation of electrical excitability in the cardiovascular system. [unreadable] [unreadable]