The general goal of this research is to elucidate mechanisms of inorganic cation transport across mitochondrial membranes. Specifically experiments will be aimed at identifying and characterizing mitochondrial K+ and MG++ transport mechanisms. Although the proposed investigations would constitute fundamental research, elucidation of mechanisms by which mitochondrial membranes control and maintain the distribution of inorganic cations between cytoplasmic and mitochondrial matrix compartments should aid in understanding deviations from steady state ion distributions which may occur in disease states. Physiological measurements have indicated that the mechanisms which mediate trans[port of K+ and Mg++ into liver mitochondria are similar in their substrate dependence, pH dependence, and sensitivity to inhibitory and stimulatory reagents. Cross competition is also observed. A protein of approximately 53 kDa has been isolated from rat liver mitochondrial membranes by affinity chromatography using immobilized quinine, an inhibitor of mitochondrial k+ and mg++ transport. Experiments will be aimed at determining whether this protein has a role in mediating K+ transport, and whether it also transports Mg++. Attempts will be made to improve the purity and yield of the 53 kDa protein. It will be reconstituted into phospholipid vesicles and planar lipid membranes, and its transport capabilities will be examined. Preliminary results indicate that the reconstituted protein or reagents known to affect K+ uniporter. Effects on transport by the reconstituted protein of reagents known to affect K+ uniporter. Effects on transport by the reconstituted protein or reagents known to affect K+ and Mg++ flux into intact mitochondria will be studied. Polyclonal antibodies prepared against the 53 kDa protein will be used to identify this protein and any related polypeptides on SDS polyacrylamide gels of mitochondrial membrane extracts. Effects of the antibodies on 42K and 28Mg flux into mitoplasts and on activities of the reconstituted 53 kDa protein will be tested.
