Mammalian mitochondria possess an inner membrane channel, the permeability transition pore (MTP), which can be inhibited by nanomolar concentrations of cyclosporin (CS) A. The molecular basis for MTP inhibition by CSA remains unclear. Mitochondria also possess a matrix cyclophilin (CyP) with a unique N-terminal sequence (CyP-M). To test the hypothesis that it interacts with the MTP, we have studied the interactions of CyP-M with rat liver mitochondria by Western blotting with a specific antibody against its unique N terminus. Although sonication in isotonic sucrose at pH 7.4 releases a large proportion of CyP-M, a sizeable CyP-M fraction sediments with submitochondrial particles at 150,000 ؋ g. We show that the interactions of this CyP-M pool with submitochondrial particles are disrupted (i) by the addition of CSA, which inhibits the pore, but not of CSH, which does not, and (ii) by acidic pH condition, which also leads to selective inhibition of the MTP; furthermore, we show that the effect of acidic pH on CyP-M binding is prevented by diethylpyrocarbonate, which fully prevents the inhibitory effect of H ؉ on the MTP (Nicolli, A., Petronilli, V., and Bernardi, P. (1993) Biochemistry 32, 4461-4465). These data suggest that CyP-M binding is involved in opening of the MTP and that pore inhibition by CSA and protons may be due to unbinding of CyP-M from its putative binding site on the MTP. A role for CyP-M in MTP regulation is also supported by a study with a series of CSA derivatives with graded affinity for CyP. We show that with each derivative the potency at inhibition of the peptidylprolyl cis-transisomerase activity of CyP-M purified to homogeneity is similar to that displayed at inhibition of MTP opening, relative to that displayed by CSA. Decreased binding to CyP-M (but not CyP-A) and decreased efficiency at MTP inhibition is obtained by substitutions in position 8 while a 4-substituted, nonimmunosuppressive derivative is as effective as the native CSA molecule, indicating that calcineurin is not involved in MTP inhibition by CSA.

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This review summarizes recent work on the regulation of the permeability transition pore, a cyclosporin A-sensitive mitochondrial channel that may play a role in intracellular calcium homeostasis and in a variety of forms of cell death. The basic bioenergetics aspects of pore modulation are discussed, with some emphasis on the links between oxidative stress and pore dysregulation as a potential cause of mitochondrial dysfunction that may be relevant to cell injury.

Energized mitochondria in sucrose medium take up a Ca2+ pulse but do not show opening of the permeability transition pore (MTP) upon membrane depolarization by uncoupler. This is due to locking of the pore in the closed conformation by matrix acidification and fast Ca2+ efflux following membrane depolarization (Petronilli, V., Cola, C., & Bernardi P. (1993) J. Biol. Chem. 268, 1011-1016). Here we show that addition of diethyl pyrocarbonate (DPC) prior to membrane depolarization restores the ability of uncoupler to induce MTP opening. Since DPC does not modify the rate and extent of matrix acidification and the rate and extent of Ca2+ release following addition of uncoupler, its effects on pore opening appear to be due to modification of histidyl residues regulating the pore open-closed probability. This hypothesis was confirmed in studies with deenergized mitochondria incubated in potassium thiocyanate medium. While at acidic pH values pore opening is otherwise prevented, DPC allows Ca2(+)-dependent pore opening at pH 6.5 in a process that maintains full sensitivity to cyclosporin A. Pore induction by DPC can be completely prevented and partially reversed by hydroxylamine, indicating that the effect of DPC can be specifically traced to carbethoxylation of histidyl residue(s) rather than to reaction with tyrosyl or sulfhydryl groups, while the possible involvement of lysyl residues cannot be excluded. Since DPC increases the pore open probability even at matrix pH values between 7.0 and 7.7, we propose that reversible protonation of one or more histidyl residues on the matrix side of the MTP plays a role in the physiological modulation of pore opening.

Survival of tumor cells is favored by mitochondrial changes that make death induction more difficult in a variety of stress conditions, such as exposure to chemotherapeutics. These changes are not fully characterized in tumor mitochondria, and include unbalance of the redox equilibrium, inhibition of permeability transition pore (PTP) opening through kinase signaling pathways and modulation of members of the Bcl-2 protein family. Here we show that a novel chemotherapeutic, the Gold(III)-dithiocarbamato complex AUL12, induces oxidative stress and tumor cell death both favoring PTP opening and activating the pro-apoptotic protein Bax of the Bcl-2 family. AUL12 inhibits the respiratory complex I and causes a rapid burst of mitochondrial superoxide levels, leading to activation of the mitochondrial fraction of GSK-3α/β and to the ensuing phosphorylation of the mitochondrial chaperone cyclophilin D, which in turn facilitates PTP opening. In addition, following AUL12 treatment, Bax interacts with active GSK-3α/β and translocates onto mitochondria, where it contributes to PTP induction and tumor cell death. These findings provide evidence that targeting the redox equilibrium maintained by mitochondria in tumor cells allows to hit crucial mechanisms that shield neoplasms from the toxicity of many anti-tumor strategies, and identify AUL12 as a promising chemotherapeutic compound.