This project will characterize three mitochondrial antiport reactions involving Na+ and K+ that are presently thought to be of physiological significance in the heart cell. These are the K+/H+, Na+/H+ and Na+/Ca2+ antiporters. It is proposed that the K+/H+ antiport participates in the maintenance of the osmotic integrity of the mitochondrion by extruding excess K+ from the matrix, and that the Na+/H+ and Na+/Ca2+ antiporters promote a cyclic influx and efflux of Na+ that results in the extrusion of mitochondrial Ca2+ and contributes to the intracellular distribution of this regulatory cation. The properties of the three antiporters will be established in isolated heart mitochondrial and in submitochondrial particles using isotope flux, ion-sensitive electrodes, the fluorescence of pH-sensitive amines, and metallochromic indicators. The temperature sensitivity, affinity for substrates, inhibitor and activator profiles, and possible regulatory features of the antiporters will be determined. This information will be used to establish assay criteria to follow these components through detergent extraction, protein fractionation, and reconstitution into liposomes. The fractionation will focus initially on Na+/Ca2+ antiport, since this component has the most characteristic inhibitor profile. All three activities will be followed in the fractions, however, and all three will ultimately be isolated in order to establish their molecular and functional properties. An additional series of protocols will establish whether or not the properties of the antiporters, as defined in intact mitochondria and in the isolated fractions, are compatible with their postulated roles in the regulation of cell metabolism.
