Increased permeability to Ca2+ in response to a depolarizing membrane potential occurs in a wide variety of cell types. In some cells this permeability increase is blocked by dihydropyridines. We propose to characterize the dihydropyridine binding site of rat cardiac tissue and to determine its subcellular location (sarcoplasmic reticulum, sarcolemma or T-tubular membranes). The first phase of this study will involve an analysis of the binding of [3H]-nitrendipine to these membranes. The calculated binding site densities in the sarcolemma will be compared to the calcium channel densities determined electrophysiologically. The binding protein will be solubilized and characterized in detergent solution. Photoaffinity derivatives of some of the calcium channel antagonists will be synthesized, assayed for activity and used, in radioactive forms, to identify and compare the specific binding proteins in the membranes. Using combined electrophoretic, chromatographic and immunological techniques, we propose to purify and characterize biochemically the antagonist binding protein. In a collaborative effort, we will use reconstitution to establish its relationship (or identify) to the voltage dependent calcium channel.
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