Mitochondrial free [Ca2+] increases during ATP/ADP antiport and ADP phosphorylation: exploration of mechanisms.

ADP influx and ADP phosphorylation may alter mitochondrial free [Ca2+] (Ca2+) and consequently mitochondrial bioenergetics by several postulated mechanisms. We tested how Ca2+ is affected by H2PO4(-) (P(i)), Mg2+, calcium uniporter activity, matrix volume changes, and the bioenergetic state. We measured Ca2+, membrane potential, redox state, matrix volume, pH(m), and O2 consumption in guinea pig heart mitochondria with or without ruthenium red, carboxyatractyloside, or oligomycin, and at several levels of Mg2+ and P(i). Energized mitochondria showed a dose-dependent increase in Ca2+ after adding CaCl2 equivalent to 20, 114, and 485 nM extramatrix free [Ca2+] (Ca2+); this uptake was attenuated at higher buffer Mg2+. Adding ADP transiently increased Ca2+ up to twofold. The ADP effect on increasing Ca2+ could be partially attributed to matrix contraction, but was little affected by ruthenium red or changes in Mg2+ or P(i). Oligomycin largely reduced the increase in Ca2+ by ADP compared to control, and Ca2+ did not return to baseline. Carboxyatractyloside prevented the ADP-induced Ca2+ increase. Adding CaCl2 had no effect on bioenergetics, except for a small increase in state 2 and state 4 respiration at 485 nM Ca2+. These data suggest that matrix ADP influx and subsequent phosphorylation increase Ca2+ largely due to the interaction of matrix Ca2+ with ATP, ADP, P(i), and cation buffering proteins in the matrix.