In a typical mammalian cell about 95% of the ATP used is produced in the mitochondria. the mitochondrion is also noted for possessing an elaborate system for transporting Ca2+. The system consists of 3 or perhaps 4 separate mechanisms: an influx mechanism (uniporter) having an extremely high Vmax, two separate efflux mechanisms, and a permeability transition which may or may not be involved in Ca2+ transport in vivo. Our long term goal is to determine the physiological role or roles which this complex system serves. A number of possible roles for this system have been suggested in the past, but only two have survived the assaults of earlier data: (1) That mitochondria control matrix [Ca2+] so as to regulate the rate of oxidative phosphorylation at least partly through control of three Ca2+-sensitive dehydrogenases, and (2) That mitochondria sequester Ca2+ in order to protect the cytosol from the effects of hypercalcemia in damaged cells. The specific aims of the current proposal are designed: (1) To test the "weak link" of the first proposed role above by determining if mitochondria can sequester Ca2+ from short pulses like those generated by hormones in vivo and if so, how the pulses' shape, intensity and periodicity affect the amount of Ca2+ taken up. (2) To evaluate Ca2+ effects on oxidative phosphorylation at intramitochondria loci other than the dehydrogenases by measuring Ca2+ effects on degrees of coupling and efficiencies of separate parts of the process. This will involve application of a new approach developed in earlier years of this grant. Some of the separate parts of the process to be studied are: generation of the electrochemical proton gradient, leakage of protons from the gradient and phosphorylation at the ATPase. (3) To evaluate a new possible role that one of the efflux mechanisms may serve a toxicological function in clearing the mitochondrion of unwanted ions preparatory to their clearance from the cell. It is becoming increasingly apparent that because of its vital role in ATP production, failure of the mitochondrial systems and loss of Ca2+ control are common irreversible steps leading to cell death. Without a better understanding of its physiological role or roles, the consequences of malfunctions of the Ca2+ transport system for the mitochondrion, the cell and the organism cannot be completely assessed.