Deciphering the mitochondrial proton leak

Abstract: Mitochondrial function underlies a broad spectrum of health and disease conditions that range from metabolic rate regulation, thermogenesis, to inborn genetic disorders, cancer, aging, cardiac and muscle dysfunction, diabetes and obesity. Using oxidative phosphorylation, the mitochondria pump protons from the matrix to the intermembrane space to generate a proton gradient. The protons return to the matrix via the ATPase complex to generate ATP that supports the life of the organism. About 20-50% of protons leak back to matrix but do not drive ATP production and this is called proton leak or uncoupling. The traditional proton leak measurement by Clark type oxygen probe and Seahorse assay use oxygen consumption rate (OCR) as a readout. These OCR based proton leak measurements cannot exclude “proton slip”, which is defined as respiratory chain activity that consumes O2 and transfers electrons without extruding protons out of the membrane. There is a knowledge gap of how to assess the specific mitochondrial proton leak and slip. To address this gap, I established a direct method for measuring the accurate proton leak and eliminating proton slip by exposing mitochondria to a pH gradient stress in saponin permeabilized cells with mt-cpYFP, a mitochondrial targeted pH sensitive indicator. The buffers lack metabolic substrates, thus making the cells energetically inactive, allowing separation of the physical property of the mitochondrial inner membrane from energetically active processes such as ion pumping. Using this approach, I have revealed that the adenine nucleotide transporter 1 (ANT1) has an under-appreciated role in supporting an excessive proton leak. With this novel mitochondrial proton leak measuring method, in this R35 MIRA proposal, we propose to 1) Compare mitochondrial proton leak between different cell types from the mt-cpYFP transgenic mouse; 2) Dissect the molecular basis of ANT1-mediated mitochondrial proton leak; 3) Screen for new drugs/compounds to prevent or augment the mitochondrial proton leak. Completion of this study will provide key mechanisms of mitochondrial proton leak and will provide a novel group of drugs that modulate mitochondrial function involved in the metabolic homeostasis of these diverse health processes.