Cooperativity of gating of ATP synthase and ant ion channels.

Abstract

Mitochondrial ATP synthase is vital not only for cellular energy production but also for energy dissipation and cell death. Our studies of highly purified, fully assembled ATP synthase monomers demonstrate that ATP synthase forms large conductance, Ca2+-sensitive and voltage-gated channels consistent with the known biophysical properties of mPTP. We have previously characterized a non-selective uncoupling channel within the ATP synthase c-subunit ring, the ATP synthase c-subunit leak channel (ACLC), the persistent opening of which initiates cell death. We have growing evidence for the role of ACLC in mitochondrial permeability transition (mPT), including reversible and irreversible gating of ACLC by the F1 as its inactivation gate. We observe dissociation of ATP synthase F1 from FO during neuronal glutamate toxicity, suggesting that non-reversible dissociation of F1 from FO occurs in pathology. However, studies of other laboratories suggest that the adenine nucleotide transporter (AAC or ANT) also forms a leak and death channel with similar biophysical characteristics to that of mPTP but different pharmacological characteristics from ACLC. Therefore, we used pharmacology to determine if ANT and ACLC cooperate in formation of the mPT pore. We isolated submitochondrial vesicles (SMVs; ATP synthasomes) from rat liver and reconstituted the SMVs with giant lipid vesicles to form larger proteoliposomes for patch clamping. “Live” antibody imaging reveals that both ANT and ATP synthase are present in the lipid vesicles. Patch clamp recordings demonstrate a multi-conductance, voltage gated channel with the biophysical and pharmacological characteristics of mPTP. Pharmacological inhibition of ANT and ATP synthase respectively demonstrates cooperative gating suggesting reciprocal interaction and/or cross-inhibition between the two channels. These findings suggest that the mPTP is comprised of regulated pores located within the ACLC and ANT, gating in a cooperative manner.