This project is designed to study mechanisms of ion transport and the factors which control membrane permeability in mitochondria. A primary objective is to characterize the K+/H+ antiporter with respect to its physiological role, the mechanism by which it is regulated by Mg++ ions and the mechanism by which it carries out electroneutral exchange of K+ for H+ across the inner membrane of mitochondria. Pharmacological inhibition of the K+/H+ antiporter and other ion transport pathways by quinine and other antimalarials will be studied in detail to determine the molecular site of action of these drugs. Development and application of quantitative thermodynamic transport equations will be required in order to characterize the ion-specific pathways which may operate simultaneously during experimental protocols. These studies will be carried out in rat liver mitochondria using ion electrodes, radioisotopes, atomic absorption spectroscopy and light scattering to measure transport. We have recently identified an 82 kDa, dicyclohexylcarbodiimide-binding protein which we believe to be responsible for K+/H+ antiport in liver mitochondria. This finding makes feasible a program designed to reconstitute a functionally active, purified K+/H+ antiporter into lipid vesicles. This program will utilize chromatography, electrophoresis and other protein fractionation procedures. K+ is the major cation of both cytosol and mitochondrial matrix. Net K+ transport into mitochondria necessarily results in matrix swelling. I have postulated that the K+/H+ antiporter is regulated through reversible inhibition by Mg++ ions and that this interaction provides a dynamic, finely tuned mechanism for matrix volume homeostasis in vivo. It is possible that the delicate balance of this K+ cycle is disturbed in pathophysiological processes. This project is designed to improve our understanding of ion transport mechanisms generally and to determine how these processes are integrated into the general energy economy of the cell.
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