Role of critical thiol groups on the matrix surface of the adenine nucleotide translocase in the mechanism of the mitochondrial permeability transition pore

Opening of the mitochondrial permeability transition pore (MPTP) is sensitized to [Ca²⁺] by oxidative stress (diamide) and phenylarsine oxide (PAO). We have proposed that both agents cross-link two thiol groups on the adenine nucleotide translocase (ANT) involved in ADP and cyclophilin-D (CyP-D) binding. Here, we demonstrate that blocking Cys¹⁶⁰ with 80μM eosin 5-maleimide (EMA) or 500μM N-ethylmaleimide (NEM) greatly decreased ADP inhibition of the MPTP. The ability of diamide, but not PAO, to block ADP inhibition of the MPTP was antagonized by treatment of mitochondria with 50μM NEM to alkylate matrix glutathione. Binding of detergent-solubilized ANT to a PAO-affinity matrix was prevented by pre-treatment of mitochondria with diamide, EMA or PAO, but not NEM. EMA binding to the ANT in submitochondrial particles (SMPs) was prevented by pre-treatment of mitochondria with either PAO or diamide, implying that both agents modify Cys¹⁶⁰. Diamide and PAO pre-treatments also inhibited binding of solubilized ANT to a glutathione S-transferase—CyP-D affinity column, both effects being blocked by 100μM EMA. Intermolecular cross-linking of adjacent ANT molecules via Cys⁵⁷ by copper phenanthroline treatment of SMPs was abolished by pre-treatment of mitochondria with diamide and PAO, but not with EMA. Our data suggest that PAO and diamide cause intramolecular cross-linking between Cys¹⁶⁰ and Cys²⁵⁷ directly (not antagonized by 50μM NEM) or using glutathione (antagonized by 50μM NEM) respectively. This cross-linking stabilizes the ‘c’ conformation of the ANT, reducing the reactivity of Cys⁵⁷, while enhancing CyP-D binding to the ANT and antagonizing ADP binding. The two effects together greatly sensitize the MPTP to [Ca²⁺].