IMAGING THE PERMEABILITY PORE TRANSITION IN SINGLE MITOCHONDRIA

In mitochondria the opening of a large proteinaceous pore, the ''mitoc hondrial permeability transition pore'' (MTP), is known to occur under conditions of oxidative stress and matrix calcium overload, MTP openi ng and the resulting cellular energy deprivation have been implicated in processes such as hypoxic cell damage, apoptosis, and neuronal exci totoxicity. Membrane potential (Delta Psi(m)) in single isolated heart mitochondria was measured by confocal microscopy with a voltage-sensi tive fluorescent dye. Measurements in mitochondrial populations reveal ed a gradual loss of Delta Psi(m) due to the light-induced generation of free radicals. In contrast, the depolarization in individual mitoch ondria was fast, sometimes causing marked oscillations of Delta Psi(m) . Rapid depolarizations were accompanied by an increased permeability of the inner mitochondrial membrane to matrix-entrapped calcein (appro ximate to 620 Da), indicating the opening of a large membrane pore. Th e MTP inhibitor cyclosporin A significantly stabilized Delta Psi(m) in single mitochondria, thereby slowing the voltage decay in averaged re cordings. We conclude that the spontaneous depolarizations were caused by repeated stochastic openings and closings of the transition pore. The data demonstrate a much more dynamic regulation of membrane permea bility at the level of a single organelle than predicted from ensemble behavior of mitochondrial populations.