Calcium transporting ATPases of the plasma membrane and intracellular membranes plays a key role in the extrusion of Ca+2 from the cell and its sequestration within intracellular organelles. Control of intracellular Ca+2 levels is of central importance in a broad range of biological processes including excitation-contraction coupling in muscle and neurotransmitter release in nerve. Recognition of the role of intracellular Ca+2 in contractility of heart muscle has contributed to an understanding of the therapeutic activity of digitalis and of drugs which interact with Ca+2-channels. A more detailed understanding of the biology of Ca+2 metabolism and of the structure and regulation of Ca+2-transporting proteins will lead to more effective strategies in the medical treatment of cardiovascular and other human diseases. The long-term objectives of this proposal are to define the structural and functional domains of Ca+2- transporting ATPases of the plasma membrane and intracellular membranes, and to describe the structure and regulation of the genes which encode these enzymes. During the past year, we have isolated and characterized cDNA clones containing the entire coding regions for two plasma membrane Ca+2 pumps and two apparent endoplasmic reticulum Ca+2 pumps. These cDNAs will be used as molecular probes in the isolation of cDNAs encoding additional Ca- ATPase isoforms expressed in heart, lung, liver and other tissues. Using Northern blot, slot blot and nuclease protection assays, we will examine the tissue distribution of the Ca-ATPase mRNAs and analyze their expression during development. The cDNAs will also be used to isolate the genes encoding these enzymes. The genes will be characterized by restriction mapping, blot hybridization and DNA sequence analysis. Potential regulatory regions will be examined by DNase footprinting and band retardation assays to identify cis regulatory elements and trans-acting factors with which they interact. The functional significance of these regions will be evaluated by examining their ability to drive the expression of a reported gene such as chloramphenicol acetyltransferase following transfection of cells in culture. In order to study the structural and functional domains of these enzymes, we will use the cDNAs in expression systems. The activity of the enzymes will be analyzed before and after modification of various domains by site-directed mutagenesis and the regions involved in functional differences between isoforms will be examined using chimeric enzymes.
