The aim of studies supported by this grant has been the elucidation of mechanisms underlying variability in cardiac repolarization, and its role in arrhythmogenesis and arrhythmia suppression. A major conceptual advance during the last period of support has been the increasing recognition that normal and abnormal electrogenesis in heart and in other excitable tissues is driven not only by pore-forming ion channel proteins, but also requires the coordinated activity of multiple other proteins, subserving functions as diverse as gating modulation, post-translational modification, trafficking, and anchoring. IKs serves a key role in maintaining normal repolarization, particularly in the face of adrenergic stimulation, and its recapitulation requires coexpression of at least KvLQT1 and minK. Preliminary data we and others have generated strongly suggest the working hypothesis that 'Ks represents a macromolecular complex that includes multiple other proteins. The overall goal of the research proposed here is testing specific hypotheses to identify' such components and define their function. We identified fhl2 as a potential minK-interacting protein in a yeast 2-hybrid assay, and have developed further in vitro data supporting an fhl2-minK interaction in heart;
in Specific Aim 1 we will extend this concept in vivo by defining the electrophysiologic phenotype of fhl2 null mice. Mistrafficking is an increasingly recognized mechanism underlying dysfunction of membrane proteins, including ion channels;
in Specific Aim 2, we will use a combination of electrophysiology, confocal imaging, and assay of cell surface protein expression -methods that are all in hand - to identify KvLQT1 domains driving normal and abnormal channel trafficking. We have identified both the PP2A phosphatase subunit B56a and the cardiac A Kinase Anchoring Protein mAXAP as potential KvLQT1 protein partners in a yeast 2-hybrid assay;
in Specific Aim 3, we will use biochemical and electrophysiologic approaches to determine interactions among channel subunits and regulatory mechanisms that specify response of 'Ks to adrenergic signaling. The results of these studies will elucidate mechanisms modulating 'Ks, further develop the tools necessary for studying how integrated multimolecular interactions modulate cardiomyocyte electrophysiology, and thereby promote the long-term goal of identifying new targets for safer, more effective antiarrhythmic intervention.
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