Functional properties of the major cell types in the cardiovascular system are regulated by a cascade of physiological and biochemical reactions beginning at the cell membrane and leading inward to the nucleus. In this Program Project, the structure, function, and regulation of cardiac forms of both sodium and potassium channels will be examined. cDNA's encoding cardiac forms of the protein components of these ion channels will be isolated and the primary structures of these proteins will be inferred from cDNA sequence. These channel proteins will be isolated from cardiac tissue and characterized. The molecular basis for their specific functional and pharmacological properties will be examined by co-expression of mRNA's encoding different combinations of subunits and by expression of mutant forms of the ion channel proteins. The regulation of these ion channels by protein phosphorylation and interaction with G proteins will also be studied by co-expression with mRNA's encoding normal and mutant forms of these regulatory proteins. Muscarinic acetylcholine receptors in cardiac cells bind acetylcholine and regulate cellular function by coupling through G proteins to adenylate cyclase, phosphatidylinositol hydrolysis, and voltage-sensitive ion channels. The molecular basis for this regulatory process will be examined by transfection of normal and mutant genes encoding specific muscarinic receptor subtypes, G proteins, and protein kinases into appropriate recipient cells and analysis of the resulting cells for receptor function and modulation. Hormonal regulation of contractile force in cardiac muscle involves activation of adenylate cyclase activity to increase intracellular levels of cAMP, activation of cAMP-dependent protein kinase, phosphorylation of the voltage-sensitive calcium channels and finally termination of the intracellular signal by hydrolysis of cAMP by cyclic nucleotide phosphodiesterases. The cDNA's encoding the cardiac form(s) of this key regulatory molecule will be isolated, their structures will be determined, and their functional properties will be examined in cellular expression experiments. A novel cGMP-inhibited form of cyclic nucleotide phosphodiesterase will be cloned and expressed and the receptor sites on this phosphodiesterase form for novel cardiotonic drugs will be studied by protein chemistry and site- directed mutagenesis. The development, function, and regulation of the cardiac cell requires the establishment and maintenance of specific functional domains. This coordinated program of investigation of cellular signal transduction in the heart will greatly enhance our understanding of the molecular basis of cardiac function and regulation.
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