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

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 A231 87. 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 Ca" 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 tertbutylhydroperoxide 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 I-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 Ca'+ 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.Keywords: mitochondria; channels ; glutathione ; pyridine nucleotide : oxidation.Mitochondria from a variety of sources possess an inner membrane channel, the permeability transition pore (MTP ; see [I, 21 for recent reviews). The MTP coincides with the mitochondrial megachannel (MMC) identified by patch-clamp experiments of rat liver mitopla...

Singlet Oxygen Produced by Photodynamic Action Causes Inactivation of the Mitochondrial Permeability Transition Pore

“…due to either energy transfers from HP excited triplet state (19) or increases in the respiratory chain e Ϫ leakage after irradiation (20), ample evidence (reviewed in Refs. 1 and 2) indicates that reactive oxygen species promote the PT, most likely through oxidation of matrix glutathione (21)(22)(23); therefore, it is unlikely that pore inhibition is mediated by other activated oxygen species, which would rather counteract the inactivating effects of 1 O 2 . From these considerations we conclude that pore inhibition in irradiated, HP-treated mitochondria most likely depends on direct effects of 1 O 2 on critical residues important for pore function.…”

“…The actual dithiol oxidant appears to be oxidized glutathione, which would mediate pore opening by a variety of oxidants (23). Cysteine is oxidized to cystine by 1 O 2 (16), and in the context of pore regulation by dithiols one could have predicted an increased probability of pore opening rather than the observed pore inactivation.…”

Mitochondria and aging: A role for the mitochondrial transition pore?

“…Oxidation–reduction of critical protein residues could also influence mPTP opening (Chernyak & Bernardi, 1996). More particularly, the oxidation of thiol functions and cysteine residues, which is an important mechanism regulating protein structure, was reported on proteins described to be involved in the formation of the pore or in the regulation of its opening, such as ANT (Costantini et al., 2000; Halestrap, Woodfield, & Connern, 1997), CypD (Nguyen et al., 2011), ATP synthase (Wang, Murray, Chung, & Van Eyk, 2013), or complex I of the respiratory chain (Chouchani et al., 2013).…”

Inhibition of the mitochondrial cyclosporin A‐sensitive permeability transition pore by the arginine reagent phenylglyoxal

“…The PT pore is controlled by several ligands as well as by the electric potential difference (A\|/) across the inner mitochondrial membrane; at high values of A\|/ the closed conformation prevails whereas a decrease in the A\|/ can trigger pore opening [4,5]. The pore is activated by Ca 2+ from the mitochondrial matrix and by an increase in the reduction potential of intramitochondrial pyridine nucleotides and glutathione [6,7].…”

“…Previous work using histidine-and cysteine-specific reagents has allowed the identification of three Abbreviations : PT, permeability transition; CsA, cyclosporin A; Ai|/, transmembrane electric potential difference; PGO, phenylglyoxal; HEPES, 4-(2-hydroxyethyl)-l-piperazine-ethanesulphonic acid; EGTA, ethylene-bis(oxoethylenenitrilo) tetraacetic acid; RR, ruthenium red; FCCP, carbonylcyanide-/>-trifluoromethoxyphenyl hydrazone separate sites that participate in the control of the PT : matrix histidine(s) conferring pH sensitivity [8]; the S-site, a dithiol which undergoes dithiol-disulfide interconversions [6,9] through matrix glutathione [7]; and the P-site, a NEM-reactive site which is in redox equilibrium with pyridine nucleotides [6,7].…”