The work proposed in this project is aimed at attaining a better understanding of the molecular mechanism of the cardiac sarcoplasmic reticulum (SR) Ca++-ATPase Calcium ions play a pivotal role in the contraction-relaxation cycle in the heart, and therefore the control and regulation of intracellular [Ca++] is a crucial factor of cardiac function. The SR Ca++-ATPase is the sole protein responsible for active transport of Ca++ into the SR. Although a large body of data exists on the kinetic aspects of the transport process, little is known concerning the conformational dynamics of energy transduction or the molecular mechanism of ion translocation. Recent developments in molecular biology have created the opportunity through site-directed mutagenesis to directly assess the role that specific amino acid residues play in enzyme specificity and catalysis. In order to utilize this powerful technique we have isolated cDNA clones for the cardiac SR Ca++-ATPase and will express it in a cell line whose viability is not dependent on Ca++-ATPase and where there is no background activity from endogenous Ca++-ATPase. This will allow us to directly test whether amino acid residues identified by chemical modification studies as being essential for Ca++ transport activity are indeed required for function. In addition, we have developed strains in which the viability of the cell is conditionally dependent on the function of the cloned enzyme which will be used to select for mutations in the Ca++-ATPase which will be generated by random mutagenesis. In conclusion, it is hoped that this study would not only bring some insight into the molecular mechanism of the SR Ca++-ATPase, but also set the stage for a more basic understanding of its role in Ca++ homeostasis in the heart.
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