Studies on the relationship between structure and function in the Ca2+ pumping ATPase of plasma membranes will be continued. These Ca2+ pumps exist in the plasma membranes of heart, smooth muscle, kidney, brain, intestinal epithelium, erythrocyte and many other cell types. They are involved in control of intracellular Ca2+ levels in cells of all kinds, and in movement of Ca2+ across epithelial cell layers such as the intestinal and kidney tubule epithelium. Information about the regulation of these pumps may thus give information relevant to hypertension and kidney disease, among other conditions. The pump consists of a single polypeptide chain of molecular weight of about 135,000 (the exact molecular weight depends on the isoform). We have succeeded in expressing this pump in COS cells and have developed an assay system for determining the activity of the expressed enzyme. We propose studies designed to extend our knowledge of the function of the different parts of this enzyme. The structure-function relationships will be mapped both by site-directed mutagenesis (utilizing the COS cell system) and by the use of synthetic peptides, whose addition modifies the activity of the pump. We will use these two techniques in combination on the same portion of the pump molecule. This coordinated use of two independent techniques will provide more reliable results, and increase our ability to verify that changes introduced into the molecule have not caused large scale alterations, in its conformation.
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