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Mitochondrial permeability transition (PT) is the phenomenon in which the mitochondrial inner membrane becomes permeable to various solutes and ions. When PT is induced by Ca(2+), cytochrome c is released from mitochondria into the cytosol where it then triggers subsequent steps of programmed cell death, apoptosis. Thus, the proteins that regulate PT and cytochrome c release could become druggable targets for various diseases. However, the mechanisms of PT and the release of cytochrome c have not yet been revealed. We previously showed that valinomycin, a potassium selective ionophore, also caused release of cytochrome c from mitochondria without inducing PT. This result indicates that cytochrome c could be released from mitochondria with or without induction of PT. In this study, to understand the difference of effects of valinomycin and Ca(2+) on mitochondria, we examined what protein species are released from valinomycin- and Ca(2+)-treated mitochondria by LC-MS/MS. As a result, only the proteins located in the intermembrane space were found to be released from valinomycin-treated mitochondria, while those in both the intermembrane space and in the matrix were released from Ca(2+)-treated mitochondria. Furthermore, the protein releases by each reagent occurred not selectively but in a concentration-dependent manner. Based on these results, the permeabilization effects of Ca(2+) and valinomycin on the inner and outer mitochondrial membranes are discussed.

Bongkrekic acid, isolated from Burkholderia cocovenenans, is known to specifically inhibit the mitochondrial ADP/ ATP carrier. However, the manner of its interaction with the carrier remains elusive. In this study, we tested the inhibitory effects of 17 bongkrekic acid analogues, derived from the intermediates obtained during its total synthesis, on the mitochondrial ATP/ ATP carrier. Rough screening of these chemicals, performed by measuring their inhibitory effects on the mitochondrial ATP synthesis, revealed that 4 of them, KH-1, KH-7, KH-16, and KH-17, had moderate inhibitory effects. Further characterization of the actions of these 4 analogues on mitochondrial function showed that KH-16 had moderate; KH-1 and KH-17, weak; and KH-7, negligible side effects of both permeabilization of the mitochondrial inner membrane and inhibition of the electron transport, indicating that only KH-7 had a specific inhibitory effect on the mitochondrial ADP/ ATP carrier. Although the parental bongkrekic acid showed a strong pH dependency of its action, the inhibitory effect of KH-7 was almost insensitive to the pH of the reaction medium, indicating the importance of the 3 carboxyl groups of bongkrekic acid for its pH-dependent action. A direct inhibitory effect of KH-7 on the mitochondrial ADP/ ATP carrier was also clearly demonstrated. [ABSTRACT FROM AUTHOR]

Yeast mitochondria have generally been believed not to undergo the permeability transition (PT) by the accumulation of Ca2+ within the mitochondrial matrix, unlike mammalian mitochondria. However, the reason why the yeast PT is not induced by Ca2+ has remained obscure. In this study, we examined in detail the effects of Ca2+ on yeast mitochondria under various conditions. As a result, we discovered that the PT could be induced even in yeast mitochondria by externally added Ca2+ under optimized experimental conditions. The 2 essential parameters for proper observation of the PT-inducing effects of Ca2+ were the concentrations of the respiratory substrate and that of inorganic phosphate (Pi) in the incubation medium. The yeast mitochondrial PT induced by Ca2+ was found to be insensitive to cyclosporin A and suppressed in the presence of a high concentration of Pi. Furthermore, when the PT was induced in yeast mitochondria by Ca2+, the release of cytochrome c from mitochondria was also observed. [Copyright &y& Elsevier]