Several properties of the myocardium are closely regulated by fluxes of Na and Ca across the sarcolemma (SL). However, insight into these processes has been difficult to obtain since study of ion transport using intact myocardial tissue is complex and rarely leads to an unequivocal interpretation. Recent biochemical advances have made it possible to begin investigations of ionic transport mechanisms with highly purified cardiac sarcolemmal vesicles. Using unique techniques, we propose to study several aspects of both sarcolemmal Na-Ca exchange and ATP-dependent Na transport in purified SL vesicles. Specifically, we are interested in the regulation of Na-Ca exchange by several factors: how different cations interact with the exchanger at both intra- and entra-cellular membrane surfaces; how the exchanger interacts with its membrane environment, especially negatively charged phosphoslipids; how the Na-Ca exchanger is affected by different Ca chelators such as EGTA and parvalbumin; how the Na- Ca exchange is effected by membrane surface potential and the diffuse double layer. Our objectives are to functionally define the operation of the exchanger, to begin to unravel its molecular characteristics, and to determine the physiologic significance of Na-Ca exchange. ATP derived from glycolysis appears to preserve membrane function and integrity better than ATP from oxidative phosphorylation during ischemia. One explanation is that membrane-bound glycolytic enzymes can directly supply ATP to membrane proteins such as the ATP-dependent Na pump. We propose to test this hypothesis using isolated SL and a new technique we have developed for measuring ATP-dependent Na transport. In red blood cells, the phospholipids, proteins, and cholesterol are distributed asymmetrically across the cell membrane. It is likely that this is also true in cardiac SL, though there is currently no supporting information available. We propose to begin investigations of the asymmetry of SL membrane components and to relate this asymmetry to the regulation of passive Ca permeability.
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