This program is concerned with the structure-function relationships and normal control mechanisms of several transport proteins and enzymes that are important in cardiac physiology, the pharmacologic regulation of these mechanisms, and their alterations under pathophysiologic conditions such as those that may result from heart failure and ischemic heart disease. These proteins include the plasma membrane NaK-ATPase, the sarcolemmal and the mitochondrial Na+/H+, K+/H+ antiporters, the mitochondrial Na+/Ca2+ antiporter, the sarcolemmal Na+, Pi-cotransporter, the cardiac protein phosphatases and their substrates, and the Ca2+-dependent proteases. The participating investigators with expertise in ion transport, membrane biochemistry, enzymology, bioenergetics, molecular biology and genetics, immunology, and cardiac pharmacology, will combine their efforts to conduct the following studies: Using cardiac sarcolemmal and mitochondrial preparations, a number of transport proteins that control the fluxes of cations and anions across the cardiac membranes will be characterized, purified, and reconstituted. The genes for these carriers will be cloned, and appropriate systems for their expressions will be developed. To study the structure-function relationships of the purified transport proteins with known sequences, alterations in protein structure will be made by mutations and chemical modifications. With the use of sealed sarcolemmal vesicles, transport proteins reconstituted in liposomes, intact mitochondria, mutants expressed in host cells, and intact cardiac myocytes, control of the functions of the various ion carriers by physiological regulators and pharmacologic agents will be studied. In studies concerned with the control of myocardial function by protein phosphorylation- dephosphorylation, characterization of cardiac protein phosphatases and their physiological substrates will be attempted, and hormonal control of these enzymes will be explored. To clarify the role of intracellular proteolytic enzymes in control of cardiac protein catabolism, interactions of Ca2+-dependent proteases with their nuclear substrates will be studied, and the role of these enzymes in the processes of membrane repair and turnover of damaged cardiac proteins will be investigated.
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