REGULATORY MECHANISMS OF CARDIAC REPOLARIZATION

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DESCRIPTION (adapted from the applicant's abstract): Voltage-gated K+ channels, responsible for action potential repolarization and setting the resting membrane potential, are proteins encoded by one of the most complex group of ion channel genes found in the cardiovascular system. A small change in K+ permeability in vascular smooth muscle membranes, results in a significant change in membrane potential and in Ca2+ channel activity. Thus, changes in K+ channel function can have an important effect on vascular tone. Three specific aims will be examined in the proposed work. 1. K+ channel subunit localization and assembly will be analyzed in rat cardiac myocytes and vascular smooth muscle tissue. Two hypotheses will be examined: (i) In heart Kva1.5 is either a homomeric alpha structure or assembled with Kva1.2 at the intercalated disk. In the ventricle (but not the atrium) the Kva1.5-containing complex is assembled with an inactivation-conferring Kvb subunit. (ii) In vascular smooth muscle, Kva1.5 exists as a heteromeric structure in association with the Kva1.4 subunit and the Kvb1.2 beta subunit. 2. The mechanisms underlying potassium channel a/b subunit interactions will be determined. Two hypotheses will be examined. (i) ab assembly involves the association of nascent channels with chaperon-like proteins that facilitate subunit assembly. (ii). Specific amino acids on b subunits determine physical ab interactions. The beta effects on voltage-sensitivity, deactivation, and inactivation can be traced to different amino acids. Furthermore all ab interactions occur at the N-terminal domains of both the alpha and beta subunits. 3. The cellular and subcellular distribution of the IKr potassium channel protein, h-erg, will be determined in normal and diseased human myocardium. One hypothesis will be investigated: IKr expression is not static, with localization being altered in diseased myocardium. The immunohistochemistry in aims 1 and 3 will be performed primarily on rat, canine and human tissue sections with antibodies that currently exist and antisera under production. Alpha-beta interactions will be monitored functionally by voltage-clamp techniques and physically by immunopurification. Subunit assembly will be determined by immunopurification methods where purification of two distinct subunits with an antibody specific for only one is the operational definition of assembly. The amino acids involved in this interaction will be identified by a variety of mutagenesis approaches.