The overall aim of the research proposed here is to determine the mechanism of Ca2+ uptake by sarcoplasmic reticulum. The primary focus of these studies is the means by which the Ca2+.Mg2+.ATPase, which is an integral part of the membrane, uses the chemical energy provided by ATP to directly transport Ca2+ across the membrane. A series of studies are designed to map out segmental movements of the ATPase and to delineate the chemical interactions which couple with enzyme conformation to control the transport mechanism. For most part, EPR spectroscopy will be used to monitor changes in interaction distances between functional sites, and follow conformational changes in the enzyme which accompany steps in the transport mechanism. Chemical modification techniques will be used to decern the role of specific residues in coupling ATP hydrolysis to transport. In turn, a variety of analytical techniques will be used to integrate the functional properties of the enzyme with observed conformational movements and decern the effects of specific modifications. Intracellular Ca2+ regulation is vital to all muscle function, including cardiac and skeletal muscle, used here for purposes of simplication. The principal role of the Ca2+.Mg2+.ATPase in this control makes the Ca2+ transport mechanism of this enzyme of primary importance. Most importantly, basic mechanisms of ion transport and energy transduction, common to many biological membranes, should be clarified by these studies.
