The Mitochondrial Permeability Transition Pore is Modulated by Oxidative Agents Through Both Pyridine Nucleotides and Glutathione at Two Separate Sites

Authors

  • Boris V. Chernyak,

    1. Consiglio Nazionale delle Ricerche Unit for the Study of Biomembranes and Laboratory of Biophysics and Membrane Biology, Department of Biomedical Sciences, University of Padova, Italy
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  • Paolo Bernardi

    Corresponding author
    1. Consiglio Nazionale delle Ricerche Unit for the Study of Biomembranes and Laboratory of Biophysics and Membrane Biology, Department of Biomedical Sciences, University of Padova, Italy
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P. Bernardi, Department of Biomedical Sciences, University of Padova, Via Trieste 75, 1-35121 Padova, Italy

Abstract

We studied the modulation of the permeability transition pore (MTP), a cyclosporin-A-sensitive channel, in deenergized mitochondria. Rat liver mitochondria were incubated in a potassium gluconate medium and treated with uncoupler, valinomycin, oligomycin and A23187. Under these conditions the membrane and Donnan potentials are collapsed, and no ion gradients are maintained, allowing the study of the dependence of MTP opening on the Ca2+ concentration under a variety of oxidative conditions without the complexities arising from changes of the membrane potential and matrix pH, and from secondary-ion redistribution.

Cross-linking of mitochondrial dithiols with arsenite or phenylarsine oxide, or treatment with tert -butylhydroperoxide leading to complete oxidation of glutathione, increased the sensitivity of MTP opening to Ca2+. This effect could be fully prevented by prior treatment of mitochondria with monobromobimane and restored by reduction with dithiothreitol. The effect of tert-butylhydroperoxide was not additive with that of AsO, and it was completely blocked by modification of reduced glutathione with 1-chloro 2,4-dinitrobenzene through glutathione-S-transferase, indicating that oxidized glutathione affects the pore through the AsO-reactive and PhAsO-reactive dithiol.

Oxidation of mitochondrial pyridine nucleotides by a variety of treatments also increased the sensitivity of MTP opening to Ca2+ under conditions where glutathione was maintained in the reduced state. This effect could be fully prevented when reduced pyridine nucleotides levels were reestablished with 2-hydroxybutyrate but not by treatment with monobromobimane or dithiothreitol.

The effects of dithiol cross-linking or oxidation, and of pyridine nucleotide oxidation on the MTP were additive. The contribution of each of these two oxidative events, when they were induced at the same time, could still be selectively blocked by monobromobimane and dithiothreitol, or by 2-hydroxybutyrate, respectively. Complete oxidation of pyridine nucleotides did not affect the reactivity of the dithiol towards monobromobimane, indicating that it remained in the reduced state.

After transient opening of the MTP, sensitivity to pyridine nucleotide oxidation was lost while sensitivity to dithiol cross-linking was retained. These data indicate that the dithiol belongs to an MTP regulatory component which is larger than the MTP cutoff of about 1500 Da, or is non-diffusible. Taken together, these findings indicate that the MTP is influenced by oxidation-reduction events at two separate sites that can be distinguished experimentally, and that these sites are not connected by common oxidation-reduction intermediates other than glutathione.

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