The major project goal is an understanding of the molecular regulation and function of cardiac sodium-calcium exchange. A secondary goal is an improved understanding of cellular sodium-calcium exchange function. The information to be gained is of basic importance to cellular physiology and is essential to an understanding of pathological cardiac function. Two unique methods will be used for molecular and cellular work, respectively; 1) current measurements in a new large-diameter sarcolemmal patch preparation, and 2) optical measurements of transmembrane calcium movements in intact tissues with new calcium dyes. In excised sarcolemmal membrane patches (>10mum diameter with >10 G-omega seals), the hypothesis will be tested that calcium activates sodium-calcium exchange by a direct mechanism, while ATP acts via a phosphorylation mechanism. The underlying mechanisms will be identified and characterized by specific biochemical interventions. Limited proteolysis by chymotrypsin, which abolishes secondary exchange modulation and leaves the exchanger highly stimulated, will allow studies of the isolated catalytic properties of sodium-calcium exchange. Relevant to the loss of secondary regulation during isolation of membrane vesicles, it will be tested whether a specific proteinase in cardiac homogenates may mimic effects of chymotrypsin and/or whether mild interventions may dissociate regulators from excised patches. In intact cardiac ventricle, fast extracellular calcium transients will be monitored with newly improved calcium-sensitive dyes and related to action potentials and contraction. Long-standing hypotheses about the physiological role of sodium-calcium exchange will be tested. The actions of inotropic mechanisms thought to act indirectly or directly via sodium- calcium exchange will be evaluated in extracellular calcium transients and related to studies of the isolated exchange current.
