NOVEL ACCESSORY PROTEIN MODULATING CARDIAC K+ CHANNELS

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Voltage-gated K+ channels (Kv) are important in the physiology of excitable and nonexcitable cells. In excitable cells, they contribute to the repolarization phase of the action potential, while in nonexcitable cells, they contribute to diverse processes such as volume regulation, hormone secretion, and activation by mitogens. K+ channels are major targets for drug treatment in a number of diseases including cardiac arrhythmias, hypertension, epilepsy, and cerebrovascular ischemia. With multiple Kv channel genes whose products may assemble as multisubunit heteromeric complexes, there may be hundreds of functionally distinct K+ channels. Given their great diversity and fundamental importance, the cellular mechanisms regulating their synthesis, assembly, and metabolism are of prime interest, but at present, almost entirely unknown. To begin to dissect these processes, we have used the yeast two-hybrid system to identify novel cytoplasmic molecules that interact with Kv channel proteins. We have cloned a novel gene encoding a Kv channel binding protein (KChAP, for K Channel Associated Protein) which modulates the expression of a subset of Kv channels in heterologous expression system assays. We hypothesize that KChAP acts as a molecular chaperone by binding transiently to Kv alpha- subunits during synthesis to promote efficient channel assembly. This proposal will focus on the characterization of the molecular mechanisms by which KChAP interacts with K+ channel subunits to enhance current expression at the cell surface. In specific aim 1, we will identify and localize KChAP-Kv channel complexes in heterologous expression systems to determine whether KChAP is attached to mature K+ channels at the cell surface, like Kv beta subunits, or whether it acts more like a true chaperone by binding transiently during early stages of channel assembly. Specific aim 2 will identify the protein domains on KChAP and Kv channels that mediate functional interactions using a combination of biochemical, electrophysiological, and immunocytochemical methods. With these techniques, we will investigate the molecular mechanisms by which KChAP increases Kv currents. The cellular location of KChAP and its relationship to Kv alpha-subunits in native tissue, with an emphasis on heart, will be pursued in specific aim 3. Finally, specific aim 4 will identify other cellular protein partners of KChAP using multiple protein interaction cloning strategies. Together these studies should provide valuable information on a novel mechanism of K+ channel regulation.