Significance: Stressful cellular events cause intracellular Ca ²⁺ dysregulation, rapid loss of inner mitochondrial membrane potential [the permeability transition (PT)], metabolic dysfunction, and death. Rapid Ca ²⁺ -induced uncoupling is one of the most important regulators of cell demise. We show that the c-subunit ring of the F ₁ F _O ATP synthase forms a voltage-sensitive channel, the persistent opening of which leads to PT and cell death. In contrast, c-subunit channel closure promotes cell survival and increased efficiency of cellular metabolism. The c-subunit channel is therefore strategically located at the center of the energy-producing complex of the cell to regulate metabolic efficiency and orchestrate the rapid onset of death and thus is a candidate for the mitochondrial PT pore.

The mitochondrial permeability transition (mPT) describes a Ca²⁺-dependent and cyclophilin D (CypD)-facilitated increase of inner mitochondrial membrane permeability that allows diffusion of molecules up to 1.5 kDa in size. It is mediated by a non-selective channel, the mitochondrial permeability transition pore (mPTP). Sustained mPTP opening causes mitochondrial swelling, which ruptures the outer mitochondrial membrane leading to subsequent apoptotic and necrotic cell death, and is implicated in a range of pathologies. However, transient mPTP opening at various sub-conductance states may contribute several physiological roles such as alterations in mitochondrial bioenergetics and rapid Ca²⁺ efflux. Since its discovery decades ago, intensive efforts have been made to identify the exact pore-forming structure of the mPT. Both the adenine nucleotide translocase (ANT) and, more recently, the mitochondrial F₁F_O (F)-ATP synthase dimers, monomers or c-subunit ring alone have been implicated. Here we share the insights of several key investigators with different perspectives who have pioneered mPT research. We critically assess proposed models for the molecular identity of the mPTP and the mechanisms underlying its opposing roles in the life and death of cells. We provide in-depth insights into current controversies, seeking to achieve a degree of consensus that will stimulate future innovative research into the nature and role of the mPTP.

Maturation of neuronal synapses is thought to involve mitochondria. Bcl-x _L protein inhibits mitochondria-mediated apoptosis but may have other functions in healthy adult neurons in which Bcl-x _L is abundant. Here, we report that overexpression of Bcl-x _L postsynaptically increases frequency and amplitude of spontaneous miniature synaptic currents in rat hippocampal neurons in culture. Bcl-x _L , overexpressed either pre or postsynaptically, increases synapse number, the number and size of synaptic vesicle clusters, and mitochondrial localization to vesicle clusters and synapses, likely accounting for the changes in miniature synaptic currents. Conversely, knockdown of Bcl-x _L or inhibiting it with ABT-737 decreases these morphological parameters. The mitochondrial fission protein, dynamin-related protein 1 (Drp1), is a GTPase known to localize to synapses and affect synaptic function and structure. The effects of Bcl-x _L appear mediated through Drp1 because overexpression of Drp1 increases synaptic markers, and overexpression of the dominant-negative dnDrp1-K38A decreases them. Furthermore, Bcl-x _L coimmunoprecipitates with Drp1 in tissue lysates, and in a recombinant system, Bcl-x _L protein stimulates GTPase activity of Drp1. These findings suggest that Bcl-x _L positively regulates Drp1 to alter mitochondrial function in a manner that stimulates synapse formation.

Purified mitochondrial ATP synthase has been shown to form Ca²⁺-activated, large conductance channel activity similar to that of mitochondrial megachannel (MMC) or mitochondrial permeability transition pore (mPTP) but the oligomeric state required for channel formation is being debated. We reconstitute purified monomeric ATP synthase from porcine heart mitochondria into small unilamellar vesicles (SUVs) with the lipid composition of mitochondrial inner membrane and analyze its oligomeric state by electron cryomicroscopy. The cryo-EM density map reveals the presence of a single ATP synthase monomer with no density seen for a second molecule tilted at an 86^o angle relative to the first. We show that this preparation of SUV-reconstituted ATP synthase monomers, when fused into giant unilamellar vesicles (GUVs), forms voltage-gated and Ca²⁺-activated channels with the key features of mPTP. Based on our findings we conclude that the ATP synthase monomer is sufficient, and dimer formation is not required, for mPTP activity.