CA TRANSPORT IN CARDIAC MEMBRANE VESICLES

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We will prepare sarcolemmal membrane vesicles from pig heart, analyze their purity and develop methods for assaying and separating vesicles of inside-out and right-side-out orientations. We will use the vesicles to study two transport processes that are thought to be important in removing Ca from myocardial cells: Na-Ca exchange and the ATP-dependent Ca pump. We will investigate the kinetic characteristics of the Na-Ca exchange system at both the cytoplasmic and extracellular membrane surfaces and evaluate the role of ATP in the operation of this transport process. We will also undertake a detailed biochemical characterization of the ATP-dependent Ca transport system, with a special emphasis on differentiating its properties from those of a similar Ca transport system in the cardiac sarcoplasmic reticulum. The possible roles of cAMP and membrane phosphorylation, and of the Ca-calmodulin system, in regulating the activity of these two transport processes will be explored. Finally, we will attempt to reconstitute these transport processes in phospholipid membranes following membrane solubilization with detergents. Reconstitution will be the first step toward the purification and molecular characterization of the membrane components responsible for transport activity.