MEASUREMENT OF MATRIX FREE MG-2+ CONCENTRATION IN RAT-HEART MITOCHONDRIA BY USING ENTRAPPED FLUORESCENT-PROBES

1. The concentration of free Mg2+ ([Mg2+](m)) within the matrix of isolated rat heart mitochondria was measured after loading of the mitochondria with the fluorescent Mg2+ indicators mag-indo-1 and mag-fura-2. No detectable change in total mitochondrial magnesium content occurred during loading with the indicators. Apparent K(d) values for Mg2+ of 3.7 mM and 2.3 mM were obtained for mag-indo-1 and mag-fura-2 respectively within mitochondria permeabilized to bivalent cations with ionomycin and the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone. These values are 2.7- and 1.8-fold greater respectively than those obtained for the free acid forms of the dyes in incubation medium. 2. Based on the above K(d) values, mitochondrial matrix Mg2+ concentrations were found to lie in the range 0.8-1.5 mM in the absence, or immediately after the addition, of a respiratory substrate. 3. Incubation of mitochondria in the presence of respiratory substrate, but in the absence of external Mg2+, led to a time-dependent decline in [Mg2+](m) to about half the initial values after 5 min. This was accompanied by a fall in the total mitochondrial magnesium content from 12.7 to 7.0 nmol/mg of protein. 4. ADP (0.5 mM), ATP (0.5 mM) or 10 mM-NaC1 had no significant effect on the fall in [Mg2+], whereas 1 μM-nigericin blocked, and 0.3 μM-valinomycin accelerated, the fall. 5. External Mg2+ concentrations above 1 mM progressively inhibited and reversed the decline in free and total mitochondrial Mg2+.