Here we define the molecular nature of the mitochondrial permeability transition pore (PTP), a key effector of cell death. The PTP is regulated by matrix cyclophilin D (CyPD), which also binds the lateral stalk of the F _O F ₁ ATP synthase. We show that CyPD binds the oligomycin sensitivity-conferring protein subunit of the enzyme at the same site as the ATP synthase inhibitor benzodiazepine 423 (Bz-423), that Bz-423 sensitizes the PTP to Ca ²⁺ like CyPD itself, and that decreasing oligomycin sensitivity-conferring protein expression by RNAi increases the sensitivity of the PTP to Ca ²⁺ . Purified dimers of the ATP synthase, which did not contain voltage-dependent anion channel or adenine nucleotide translocator, were reconstituted into lipid bilayers. In the presence of Ca ²⁺ , addition of Bz-423 triggered opening of a channel with currents that were typical of the mitochondrial megachannel, which is the PTP electrophysiological equivalent. Channel openings were inhibited by the ATP synthase inhibitor AMP-PNP (γ-imino ATP, a nonhydrolyzable ATP analog) and Mg ²⁺ /ADP. These results indicate that the PTP forms from dimers of the ATP synthase.

Blue native gel electrophoresis purification and immunoprecipitation of F(0)F(1)-ATP synthase from bovine heart mitochondria revealed that cyclophilin (CyP) D associates to the complex. Treatment of intact mitochondria with the membrane-permeable bifunctional reagent dimethyl 3,3-dithiobis-propionimidate (DTBP) cross-linked CyPD with the lateral stalk of ATP synthase, whereas no interactions with F(1) sector subunits, the ATP synthase natural inhibitor protein IF1, and the ATP/ADP carrier were observed. The ATP synthase-CyPD interactions have functional consequences on enzyme catalysis and are modulated by phosphate (increased CyPD binding and decreased enzyme activity) and cyclosporin (Cs) A (decreased CyPD binding and increased enzyme activity). Treatment of MgATP submitochondrial particles or intact mitochondria with CsA displaced CyPD from membranes and activated both hydrolysis and synthesis of ATP sustained by the enzyme. No effect of CsA was detected in CyPD-null mitochondria, which displayed a higher specific activity of the ATP synthase than wild-type mitochondria. Modulation by CyPD binding appears to be independent of IF1, whose association to ATP synthase was not affected by CsA treatment. These findings demonstrate that CyPD association to the lateral stalk of ATP synthase modulates the activity of the complex.

The oligomycin-sensitivity conferring protein (OSCP) of the mitochondrial F_OF₁ ATP synthase has long been recognized to be essential for the coupling of proton transport to ATP synthesis. Located on top of the catalytic F₁ sector, it makes stable contacts with both F₁ and the peripheral stalk, ensuring the structural and functional coupling between F_O and F₁, which is disrupted by the antibiotic, oligomycin. Recent data have established that OSCP is the binding target of cyclophilin (CyP) D, a well-characterized inducer of the mitochondrial permeability transition pore (PTP), whose opening can precipitate cell death. CyPD binding affects ATP synthase activity, and most importantly, it decreases the threshold matrix Ca²⁺ required for PTP opening, in striking analogy with benzodiazepine 423, an apoptosis-inducing agent that also binds OSCP. These findings are consistent with the demonstration that dimers of ATP synthase generate Ca²⁺-dependent currents with features indistinguishable from those of the PTP and suggest that ATP synthase is directly involved in PTP formation, although the underlying mechanism remains to be established. In this scenario, OSCP appears to play a fundamental role, sensing the signal(s) that switches the enzyme of life in a channel able to precipitate cell death.

The permeability transition pore (PTP) is a latent, high‐conductance channel of the inner mitochondrial membrane. When activated, it plays a key role in cell death and therefore in several diseases. The investigation of the PTP took an unexpected turn after the discovery that cyclophilin D (the target of the PTP inhibitory effect of cyclosporin A) binds to F_OF₁ (F)‐ATP synthase, thus inhibiting its catalytic activity by about 30%. This observation was followed by the demonstration that binding occurs at a particular subunit of the enzyme, the oligomycin sensitivity conferral protein (OSCP), and that F‐ATP synthase can form Ca²⁺‐activated, high‐conductance channels with features matching those of the PTP, suggesting that the latter originates from a conformational change in F‐ATP synthase. This review is specifically focused on the OSCP subunit of F‐ATP synthase, whose unique features make it a potential pharmacological target both for modulation of F‐ATP synthase and its transition to a pore.Linked Articles: This article is part of a themed section on Mitochondrial Pharmacology: Featured Mechanisms and Approaches for Therapy Translation. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.22/issuetoc