The goal of this proposal is to study the importance of sarcolemmal calcium transport mechanisms, the Na/Ca exchange mechanism and the Ca-ATPase, in regulating the intracellular Ca2+ homeostasis of the cardiac cell. The proposed experiments will examine 1) under what conditions the Na/Ca exchange mechanism controls """"""""resting"""""""" or steady state [Ca2+], 2) how Na/Ca exchange regulates the size of the stimulate [Ca2=}, transient and 3) the role of the Ca-ATPase in controlling [Ca2+], homeostasis in cardiac cells, experiments will focus on a detailed kinetic analysis of the Ca2+ flux via Na/Ca exchange and the Ca2+ extrusion by the Ca-ATPase. To accomplish these objectives, experiments will be performed on single cardiac ventricular myocytes which are voltage clamped with a low resistance patch pipette. The patch electrode will contain fluorescent indicator for measurement of Ca2+, Na+ or H+ as well as specific concentrations of various ions (Na+, for example) and Ca2+ buffers so that ionic gradients across the cell membrane and cellular Ca2+ buffering can be controlled. This experimental technique will allow for simultaneous measurement of membrane currents, intracellular ion concentration and membrane Ca2+ transport. In this way, the amount of Ca2+ flux via the Na/Ca exchange mechanism and the Ca2+-ATPase can be accurately determined under a variety of conditions. Because the Na/Ca exchanger and the Ca-ATPase are responsible for maintaining Ca2+ homeostasis within the cardiac cell, they play a critical role in controlling cell contraction and the underlying Ca2+ Transient. These sarcolemmal transport mechanisms may be particularly important under pathological conditions when changes in [Ca2+], in response to increases in intracellular Na+ and H+ concentrations may determine whether electrical and mechanical disturbances will occur.
