REGULATION OF THE CARDIAC NA+/CA++ EXCHANGER

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The overall objective of this project is to define the molecular mechanism of regulation of the cardiac Na/Ca exchange system. The primary hypothesis to be tested is that regulation of exchange activity involves specific regions within the central hydrophilic domain of the exchange carrier. The Na/Ca exchange system is a plasma membrane carrier-mediated transport process which couples the movement of 3 Na ions in one direction to the movement of a single Ca ion in the opposite direction. It is the principal Ca efflux process in cardiac myocytes and plays a major role in regulating cardiac contractility. The cloned bovine cardiac Na/Ca exchanger has been permanently expressed in CHO cells and the regulatory behavior of exchange activity in these cells will be characterized with respect to (a) its dependence upon cellular ATP levels and intracellular Ca, (b) the influence of protein kinases and phosphatases on activity and (c) the possible involvement of aminophospholipid translocase activity in mediating ATP-dependent regulation. To define regions of the hydrophilic domain that are involved in the regulation of transport activity, a series of deletion mutants will be prepared and their regulatory and functional properties will be examined by transient expression in COS cells. Finally, specific regions within the hydrophilic domain that might be involved in the regulation of exchange activity will be altered by site-directed mutagenesis and the regulatory behavior of these mutants will be examine in transfected COS and/or CHO cells. Understanding the mechanisms involved in regulating Na/Ca exchange activity will provide important insights into the regulation of cardiac contractility and the pathophysiology of cardiac failure.