Mitochondrial Megachannel Resides in Monomeric ATP Synthase

Abstract

Mitochondrial ATP synthase has been shown recently to be vital not only for cellular energy production but also for energy dissipation and cell death. We identified and characterized a large non-selective uncoupling channel within the ATP synthase c-subunit ring, the persistent opening of which initiates cell death. We have continuing evidence for the crucial role of this channel in mitochondrial permeability transition (mPT). We have now purified ATP synthase from porcine heart mitochondria and performed single-channel studies. Excised proteoliposome patch-clamp recordings demonstrate that highly pure and fully assembled ATP synthase monomers form large conductance, Ca2+-sensitive and voltage-gated channels. We confirmed the monomeric state of ATP synthase by cryo-electron microscopy studies of ATP synthase reconstituted into proteoliposomes. We have also heterologously overexpressed and purified human ATP synthase c-subunit from E. coli plasma membranes. We show that human c-subunit purified from bacteria forms large conductance channels identical to those purified from HEK-293 cells. The channel is gated by polar amino acid residues situated at the mouth of the pore and by the hydrophilic F1 portion of ATP synthase. We find that dissociation of ATP synthase F1 from FO occurs when we expose primary hippocampal neurons to glutamate toxicity, suggesting that the non-reversible dissociation of F1 from FO is pathological. We have successfully knocked out five of the six alleles of the three genes encoding ATP synthase c-subunit in mouse embryonic stem cells by CRISPR-Cas9, which resulted in only 10% of the total c-subunit expression. Patch-clamp recordings of mitoplasts isolated from these cells demonstrate low conductance activity that is poorly calcium responsive. These findings confirm that the largest of all inner mitochondrial membrane conductances resides within the ATP synthase monomer, more specifically within its membrane-embedded c-subunit ring.