Patients with heart disease are frequently exposed to an abnormal and dynamic neurohormonal milieu, with resultant varying activation of cellular signalling systems. Available data indicate that most cardiac ion channels in native myocytes are regulated by cellular signalling systems. However, analysis of specific currents is complicated by the presence of multiple components most likely representing structurally distinct ion channels. This Project will combine electrophysiologic and biochemical methods to investigate the mechanisms by which cellular metabolic processes regulate the function of individual cardiac potassium channels. Cloned human and rat potassium channels expressed in a mammalian tissue culture system will be studied using whole-cell techniques to determine if stimulation of protein kinase A or C or alterations in intracellular calcium alter outward current; more detailed experiments using a range of modulators, including catalytic subunits of individual kinases, in excised and cell-attached patches will determine the electrophysiologic mechanisms of such modulation. Parallel studies will be conducted in native myocytes displaying outward current resembling that of the clones. Bacterially synthesized fusion proteins will be used to directly determine the extent to which cloned channel proteins can serve as substrates for phosphorylation by protein kinase A and C, and specific phosphorylated residues will be mapped using HPLC and mass spectrometry. Amino acids identified as phosphorylation sites will be mutated and the functional consequences for the electrophysiology and response to cellular regulation of the mutant protein determined. The effects of ion channel blockers on metabolically stimulated and unstimulated channels will be compared to determine if response to blockers and to stimulation is additive, or if response to blockers is qualitatively altered in stimulated systems. These studies will determine mechanisms and functional consequences of ion channel phosphorylation and may thereby identify circumstances in which arrhythmia provocation or altered antiarrhythmic drug effect occur in patients.
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