The cardiac plasma membrane or sarcolemma (SL) regulates ion flux into and out of the cardiac cell through a series of channels, pumps and antiporters. The control of transmembrane ion flux is necessary not only for cellular homeostasis, but also as the means by which hormones and neurotransmitters influence cardiac contractile and electrical function. The ways in which an individual cell controls these ion transport activities are not well understood. Evidence from numerous systems suggests that phosphorylation of membrane proteins may be an important control mechanism for transmembrane ion movement. We propose to investigate the phosphorylation of a novel membrane protein localized to cardiac SL and its effects on the transport of ions across purified cardiac SL vesicles. This protein of molecular weight 15,000 (15-kDa protein) is phosphorylated in both cardiac SL vesicles and in intact functioning myocardium in response to a number of biochemical and hormonal stimuli including both beta- and alpha-adrenergic stimulation. It is a unique substrate of both cAMP-dependent protein kinase and protein kinase C in purified SL vesicles. Preliminary data by ourselves and others suggest that the 15-kDa protein may regulate either SL Ca channels and/or sodium-proton exchange. Using state-of-the-art biochemical techniques we plan to: 1) purify the 15-kDa protein to homogeneity, 2) determine its amino acid sequence and localize the sites of phosphorylation, 3) generate monoclonal and polyclonal antibodies to the 15-kDa protein as aids for purification and as probes to determine its precise subcellular localization and function, and 4) by biochemical and electrophysiologic experiments determine the function(s) of the 15-kDa protein in the regulation of cardiac SL ion transport. The program will develop the skill of the candidate in the preparation of purified cardiac SL vesicles, the isolation, purification and biochemical characterization of membrane proteins, and the techniques necessary to study ion transport at the subcellular and purified protein levels. The long term objective of the candidate is to apply the tools of biochemistry to understanding the control of cellular electrophysiology in physiologic and pathophysiologic states.
