(35) REPETITIVE GATING OF THE PERMEABILITY TRANSITION PORE IN SINGLE MITOCHONDRIA IN VITRO AND IN THE LIVING CELL

Jörg Hüser and Lothar A. Blatter
Department of Physiology and The Cardiovascular Institute, Loyola University Chicago, Maywood, IL 60153

Today it is generally accepted that the Ca-induced mitochondrial permeability increase is caused by opening of a large conductance pore residing in the inner membrane, i.e. the permeability transition pore (PTP). The permeability transition has been extensively studied in suspensions of isolated organelles. In population measurements the transition is apparent as a gradual process, e.g. loss of membrane potential or matrix swelling, reflecting the successive recruitment of individual organelles to undergo pore opening. In contrast, in a single mitochondrion PTP opening is instantaneous, leading to rapid dissipation of electrical and ionic gradients. However, because of signal averaging over large populations important information on the kinetics of PTP is not resolvable in this type of experiment.

We have used isolated cardiac mitochondria and confocal imaging of the potentiometric fluorescent probe tetramethylrhodamine ethyl ester (TMRE) to measure changes in membrane potential in single organelles. The recordings revealed rapid spontaneous depolarizations caused by opening(s) of PTP. Moreover, repetitive openings and closings of the pore resulted in marked fluctuations of membrane potential between the fully energized and the completely discharged (FCCP-insensitive) state. Under our experimental conditions pore opening was triggered by the photo-induced generation of oxygen radicals and the subsequent oxidation of protein thiols. Moreover, prolonged laser illumination of TMRE-stained mitochondria in living vascular endothelial cells resulted in membrane potential fluctuations similar to those observed in isolated organelles.

In intact cardiac myocytes stained with TMRE illumination with high intensity laser light resulted in abrupt loss of dye from individual mitochondria. The depolarization was not synchronized among individual mitochondria in a single cell. Fluorescence decay was fast in the single mitochondrion suggesting the involvement of PTP to cause rapid depolarization. The light-induced depolarization, however, was unaffected by cyclosporin A. When the excitation energy was reduced mitochondrial membrane permeability was restored and mitochondria recharged. During the recovery process we observed: (i) large voltage fluctuations in single organelles or very small groups of mitochondria; (ii) simultaneous recovery of membrane potential in chains of interfibrillar mitochondria stretching over up to 10 sarcomeres (20 m) suggesting electrical coupling between neighboring organelles (see Amchenkova et al. J. Cell Biol. 107:481, 1988); and (iii) propagation (1-2 m/s) of the repolarization front in a wave-like fashion similar to the propagated redox-waves in substrate-deprived heart cells recently reported by Romashko et al. (Proc. Natl. Acad. Sci. USA 95:1618, 1998). These results indicate a dynamic and coordinated organization of the oxidative energy metabolism at the subcellular level in cardiac myocytes.