Fluctuations in mitochondrial membrane potential caused by repetitive gating of the permeability transition pore

Confocal laser scanning microscopy and the potentiometric fluorescence probe tetramethylrhodamine ethyl ester were used to measure changes in membrane electrical potential (Delta Psi(m)) in individual mitochondria after isolation or in the living cell. Recordings averaged over small mitochondrial populations revealed a gradual decline in Delta Psi(m) caused by the light-induced generation of free radicals. Depolarization was attenuated by dithiothreitol or acidification. In contrast, individual organelles displayed rapid spontaneous depolarizations caused by openings of the mitochondrial permeability transition pore (MTP). Repetitive openings and closings of the pore gave rise to marked fluctuations in Delta Psi(m) between the fully charged and completely depolarized state. Rapid spontaneous fluctuations in Delta Psi(m) were observed in mitochondria isolated from rat heart and in mitochondria in living endothelial cells. The loss of Delta Psi(m) of mitochondria in the living cell coincided with swelling of the organelle and the breakdown of long mitochondrial filaments. In the individual mitochondrion, oxidative stress initially triggered pore openings of shorter duration, before prolonged openings caused the complete dissipation of Delta Psi(m) and a measurable efflux of larger solutes. Generalizing this scheme, we suggest that under conditions of prolonged oxidative stress and/or cellular Ca2+ overload, short openings of MTP might serve as an emergency mechanism allowing the partial dissipation of Delta Psi(m), the fast release of accumulated Ca2+ ions and the decreased generation of endogenous oxygen radicals. In contrast, loss of matrix metabolites, swelling and other structural damage of the organelle render prolonged openings of the transition pore deleterious to mitochondria and to the cell.