Modulation of the mitochondrial permeability transition pore by pyridine nucleotides and dithiol oxidation at two separate sites.
After accumulation of a Ca2+ load, the addition of uncoupler to respiring rat liver mitochondria is followed by opening of the permeability transition pore (MTP), a voltage-dependent channel sensitive to cyclosporin A. The channel's voltage threshold is profoundly affected under conditions of oxidative stress, with a shift to more negative values that may cause MTP opening at physiological membrane potentials. In this paper we further clarify the mechanisms by which oxidative agents affect the apparent voltage dependence of the MTP. We show that two sites can be experimentally distinguished. (i) A first site is in apparent oxidation-reduction equilibrium with the pyridine nucleotide (PN) pool (NADH/NAD + NADPH/NADP); PN oxidation is matched by increased MTP open probability under conditions where the glutathione pool is kept in the fully reduced state; this site can be blocked by N-ethylmaleimide but not by monobromobimane, a thiol-selective reagent. (ii) A second site coincides with the oxidation-reduction-sensitive dithiol we have recently identified (Petronilli, V., Costantini, P., Scorrano, L., Colonna, R., Passamonti, S., and Bernardi, P. (1994) J. Biol. Chem. 269, 16638-16642); dithiol cross-linking at this site by arsenite or phenylarsine oxide is matched by increased MTP open probability under conditions where the PN pool is kept in the fully reduced state; at variance from the first, this site can be blocked by both N-ethylmaleimide and monobromobimane and is probably in equilibrium with the glutathione pool. Based on these findings, we reassess the mechanisms by which many oxidative agents affect the MTP and resolve conflicting reports on the relative role of PN and glutathione oxidation in the permeability transition within the framework of MTP (dys)regulation at two separate sites.
Costantini
P
P
Consiglio Nazionale delle Ricerche Unit for the Study of Physiology of Mitochondria, University of Padova Medical School, Via Trieste 75, I-35121 Padova, Italy.
Chernyak
B V
BV
Petronilli
V
V
Bernardi
P
P
D020155:3-Hydroxybutyric Acid; D000090:Acetoacetates / Q000494:pharmacology; D000818:Animals; D001152:Arsenicals / Q000494:pharmacology; D001643:Bridged Bicyclo Compounds / Q000494:pharmacology; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone / Q000494:pharmacology; D005978:Glutathione / Q000378:metabolism; D006885:Hydroxybutyrates / Q000494:pharmacology; D007425:Intracellular Membranes / Q000187:drug effects / Q000378:metabolism*; D015640:Ion Channel Gating / Q000187:drug effects; D008930:Mitochondria, Liver / Q000187:drug effects / Q000378:metabolism*; D009243:NAD / Q000378:metabolism*; D009249:NADP / Q000378:metabolism*; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D010539:Permeability / Q000187:drug effects; D051381:Rats; D012402:Rotenone / Q000494:pharmacology; D013438:Sulfhydryl Compounds / Q000378:metabolism*
D000090:Acetoacetates; D001152:Arsenicals; D001643:Bridged Bicyclo Compounds; D006885:Hydroxybutyrates; D013438:Sulfhydryl Compounds; D012402:Rotenone; C029341:oxophenylarsine; D009243:NAD; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; C016635:acetoacetic acid; D009249:NADP; D005978:Glutathione; D020155:3-Hydroxybutyric Acid; C028381:monobromobimane