Use of dyes to estimate the electrical potential of the mitochondrial membrane

A number of cationic or anionic fluorescent dyes were investigated as possible monitors of the membrane potential of rat liver mitochondria, and giant mitochondria isolated from the liver of mice maintained on a diet containing cuprizone. The fluorescence of four dyes (8-anilino-1-naphthalenesulfonic acid, merocyanine 540, 3,3'-dipropyl-thiocarbocyanine, and bis[1,3-dibutylbarbituric acid-(5)]-pentamethine oxonol) was found to respond appropriately to changes in an apparent K+ diffusion potential. Generally, valinomycin-induced K+ diffusion potentials as calculated using the Nernst equation were used to calibrate the dependence of the fluorescence on the membrane potential. The appropriateness of this approach was verified for two dyes using microelectrodes in giant mitochondria. The apparent membrane potential change induced by the addition of succinate was variable but was very low and generally less than 60 mV in magnitude. The results are consistent with the notion that a large membrane potential is not established upon the initiation of metabolism and that the membrane potential does not play a significant role in the observed ADP phosphorylation.