Repetitive Transient Depolarizations of the Inner Mitochondrial Membrane Induced by Proton Pumping
Single mitochondria show the spontaneous fluctuations of ΔΨm. In this study, to examine the mechanism of the fluctuations, we observed ΔΨm in single isolated heart mitochondria using time-resolved fluorescence microscopy. Addition of malate, succinate, or ascorbate plus TMPD to mitochondria induced...
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Published in | Biophysical journal Vol. 88; no. 3; pp. 2340 - 2349 |
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Main Authors | , , , , |
Format | Journal Article |
Language | English |
Published |
United States
Elsevier Inc
01.03.2005
Biophysical Society |
Subjects | |
Online Access | Get full text |
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Summary: | Single mitochondria show the spontaneous fluctuations of ΔΨm. In this study, to examine the mechanism of the fluctuations, we observed ΔΨm in single isolated heart mitochondria using time-resolved fluorescence microscopy. Addition of malate, succinate, or ascorbate plus TMPD to mitochondria induced polarization of the inner membrane followed by repeated cycles of rapid depolarizations and immediate repolarizations. ADP significantly decreased the frequency of the rapid depolarizations, but the ADP effect was counteracted by oligomycin. On the other hand, the rapid depolarizations did not occur when mitochondria were polarized by the efflux of K+ from the matrix. The rapid depolarizations became frequent with the increase in the substrate concentration or pH of the buffer. These results suggest that the rapid depolarizations depend on the net translocation of protons from the matrix. The frequency of the rapid depolarizations was not affected by ROS scavengers, Ca2+, CsA, or BA. In addition, the obvious increase in the permeability of the inner membrane to calcein (MW 623) that was entrapped in the matrix was not observed upon the transient depolarization. The mechanisms of the spontaneous oscillations of ΔΨm are discussed in relation to the matrix pH and the permeability transitions. |
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Bibliography: | ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 Address reprint requests to Yoshihiro Ohta, E-mail: ohta@cc.tuat.ac.jp. Abbreviations used: ΔΨm, mitochondrial membrane potential; ANT, adenine nucleotide translocator; BA, bongkrekic acid; BCA, bicinchoninic acid; BSA, bovine serum albumin; calcein-AM, calcein acetoxymethyl ester; CsA, cyclosporin A; DTT, dithiothreitol; GSH, reduced glutathione; IMAC, mitochondrial inner membrane anion channel; MPT, mitochondrial permeability transition; ROS, reactive oxygen species; TMPD, tetramethyl-p-phenylenediamine; TMPyP, Mn(III)tetrakis(1-methyl-4-pyridil)porphyrin pentachloride. |
ISSN: | 0006-3495 1542-0086 |
DOI: | 10.1529/biophysj.104.041483 |