Biophysical Journal
Volume 88, Issue 3, March 2005, Pages 2340-2349
Journal home page for Biophysical Journal

Repetitive Transient Depolarizations of the Inner Mitochondrial Membrane Induced by Proton Pumping

https://doi.org/10.1529/biophysj.104.041483Get rights and content
Under an Elsevier user license
open archive

Abstract

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.

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

Cited by (0)