Atrial fibrillation is the most common sustained cardiac arrhythmia in the United States, and it remains a major source of morbidity and mortality in this country. Antiarrhythmic drugs currently available to treat this arrhythmia are often ineffective, and they can create serious proarrhythmia because ion channels in the ventricle are affected. The goal of this proposal is to investigate the molecular basis of an atrial-specific ultrarapid K+ current, IKur, a potential target for pharmacologic therapy of atrial fibrillation. While the Kv 1.5 gene product is an important component of IKur, our preliminary data indicate that this alpha-subunit cannot fully recapitulate the native K+ current. In the proposed specific aims, we will test the hypothesis that IKur is a macromolecular complex composed of multiple channel subunits, signaling molecules, and additional proteins that can modify channel function. The Kv 1.5 complex will be isolated from human atrium, and associated K+ channel alpha and/or beta-subunits will be identified using antibody-based methods. Following heterologous expression of the proteins identified, electrophysiologic techniques will be used to confirm if the resultant K+ current phenotype is that of IKur. Additional experiments will determine whether chamber and disease-specific alterations in the channel complex occur. An analogous strategy will be used to determine the role of A-kinase anchoring proteins (AKAPs) in the Kv 1.5 signaling complex. We will also test the hypothesis generated by our preliminary data that a Kv beta subunit can function as an AKAP. Finally, a proteomics approach will be employed to identify previously unknown protein partners in the Kv 1.5 complex, with protein-protein interactions validated using standard biochemical approaches. This technology will also be used to confirm associated Kv subunits and signaling molecules. The knowledge gained from these studies will improve our understanding of the molecular components of atrial electrophysiology and facilitate the development of novel strategies in the treatment of atrial fibrillation.