The objective of this proposal is to isolate and characterize three monovalent cation transporters of physiological importance in heart mitochondria. These are (a) the Na+/H+ antiport and (b) the Na+/Ca2+ antiport which, acting in concert contribute to mitochondrial Ca2+ homeostasis and the regulation of metabolic activity by providing a pathway for Ca2+ efflux from the matrix and (c) the latent K+/H+ antiport, which is thought to provide the mitochondrion with a cation- extruding mechanism for maintaining matrix volume. The mitochondrial monovalent cation-extruding mechanism for maintaining matrix volume. The mitochondrial monovalent cation antiports share some properties with the corresponding activities found in microbial membranes and those in the plasma membrane of eukaryotes, but show significant differences in others. All three of these transporters react with Na+ and this overlapping substrate specificity combined with the lack of suitable affinity probes makes it necessary to establish adequate inhibitor and kinetic criteria for their rigorous identification during purification and reconstitution protocols. The symmetry of the Na+/H= antiport will be examined by establishing the kinetic properties of Na+ influx, Na+ efflux and Na+/Na+ exchange in intact mitochondria and influx into submitochondrial particles (SMP). The Na+-binding features of the Na+/H+ and Na+/C2+ antiport will be compared by establishing the sensitivity of each to amiloride analogues and the location of amiloride-binding sites. The effect of matrix pH and pCa on Na+/Ca2+ antiport will be determined in tact mitochondria and SMP. The kinetic properties of the Mg2+ - sensitive K+/H+ antiport will be examined by following the extrusion of matrix 42K+ and steady-state K+/K+ exchange. These techniques will also be used to estimate the turnover of matrix K+ under defined metabolic conditions in order to test the concept that this antiport functions as an osmotic defense mechanism. Reliable assay criteria will be developed for each of the 3 antiports in reconstitution protocols. These assays will then be used to solubilize the transporters in detergents, purify the solubilized proteins by chromatography on hydroxylapatite and other supports and reconstitute the active fractions into proteoliposomes. These active purified antiports will then be available for studies relating the activity of these proteins to their structure.
