Rhodamine 123 as a probe of mitochondrial membrane potential: evaluation of proton flux through F(0) during ATP synthesis.

Rhodamine 123 (RH-123) was used to monitor the membrane potential of mitochondria isolated from rat liver. Mitochondrial energization induces quenching of RH-123 fluorescence and the rate of fluorescence decay is proportional to the mitochondrial membrane potential. Exploiting the kinetics of RH-123 fluorescence quenching in the presence of succinate and ADP, when protons are both pumped out of the matrix driven by the respiratory chain complexes and allowed to diffuse back into the matrix through ATP synthase during ATP synthesis, we could obtain an overall quenching rate proportional to the steady-state membrane potential under state 3 condition. We measured the kinetics of fluorescence quenching by adding succinate and ADP in the absence and presence of oligomycin, which abolishes the ADP-driven potential decrease due to the back-flow of protons through the ATP synthase channel, F(0). As expected, the initial rate of quenching was significantly increased in the presence of oligomycin, and conversely preincubation with subsaturating concentrations of the uncoupler carbonyl cyanide p-trifluoro-metoxyphenilhydrazone (FCCP) induced a decreased rate of quenching. N,N'-dicyclohexylcarbodiimide (DCCD) behaved similarly to oligomycin in increasing the rate of quenching. These findings indicate that RH-123 fluorescence quenching kinetics give reliable and sensitive evaluation of mitochondrial membrane potential, complementing steady-state fluorescence measurements, and provide a mean to study proton flow from the mitochondrial intermembrane space to the matrix through the F(0) channel.