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Fluctuations in Mitochondrial Membrane Potential in Single Isolated Brain Mitochondria: Modulation by Adenine Nucleotides and Ca²⁺

Department of Pharmacology, University of Pittsburgh, Pittsburgh, Pennsylvania

Correspondence: Address reprint requests to Ian J. Reynolds, Dept. of Pharmacology, University of Pittsburgh, W1351 Biomedical Science Tower, Pittsburgh, PA 15261. Tel.: 412-648-2134; Fax: 412-624-0794; E-mail: iannmda{at}pitt.edu.

In this study we investigated fluctuations in mitochondrial membrane potential (_m) in single isolated brain mitochondria using fluorescence imaging. Mitochondria were attached to coverslips and perfused with K⁺-based buffer containing 20 µM EDTA, supplemented with malate and glutamate, and rhodamine 123 for _m determination. _m fluctuations were triggered by mitochondrial Ca²⁺ uptake since they were inhibited by both ruthenium red, a Ca²⁺-uniporter blocker, and by high concentrations of EGTA. A very low concentration of Ca²⁺ (30 nM) was required to initiate the fluctuations. Both ATP and ADP reversibly inhibited _m fluctuations, with maximal effects occurring at 100µM. The effect of nucleotides could not be explained by the reversed mode of mitochondrial ATP-synthase, since oligomycin was not effective and nonhydrolysable analogs of ATP and ADP did not stop the fluctuations. The effects of adenine nucleotides were abolished by blockade of the adenine nucleotide translocator with carboxyatractyloside, but were insensitive to another inhibitor, bongkrekic acid. ATP-sensitive K⁺-channels are not involved in the mechanism of _m fluctuations, since the inhibitor 5-hydroxydecanoate or the activator diazoxide did not affect dynamics of _m. We suggest _m fluctuations in brain mitochondria are not spontaneous, but are triggered by Ca²⁺ and are modulated by adenine nucleotides, possibly from the matrix side of the inner mitochondrial membrane.

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