Repetitive transient depolarizations of the inner mitochondrial membrane induced by proton pumping

¹ Tokyo University of Agriculture and Technology

^* To whom correspondence should be addressed. E-mail: ohta{at}cc.tuat.ac.jp.

Submitted on February 14, 2004
Revised on March 25, 2004
Accepted on 30 December 2004

	Abstract

Single mitochondria show the spontaneous fluctuations of m. In this study, to examine the mechanism of the fluctuations, we have observed m in single isolated heart mitochondria using time-resolved fluorescence microscopy. Addition of malate, succinate or ascorbate plus tetramethyl-p-phenylenediamine 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+, cyclosporin A or bongkrekic acid. 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 m are discussed in relation to the matrix pH and the permeability transitions.

Key Words: electron transfer chain, membrane potential, mitochondria, permeability transition, proton pump