Molecular Biophysics of Mitochondrial Membranes

Project summary Peter Mitchell’s chemiosmotic theory postulated that the inner mitochondrial membrane (IMM) must have very low conductance for physiological ions to prevent dissipation of the voltage across the IMM (ΔΨ), which is used by ATP synthase to generate ATP. Interestingly, studies conducted in the 1960s and 1970s suggested that, contrary to the chemiosmotic theory, the IMM does conduct K+ and Cl-. Since then, there has been a significant push to identify and characterize the proteins responsible for these conductances, as their existence in the IMM despite possible ΔΨ dissipation suggests crucial physiological roles. However, even after many decades, the molecular identity, significance, and basic functional properties of the K+ and Cl- conductances of the IMM remain elusive. A major barrier to understanding these conductances was the inability to apply direct electrophysiological methods for their functional identification and characterization. This limitation led to the exclusive use of indirect methods, which produced conflicting results and caused significant confusion. In this project, we successfully applied whole-IMM patch-clamp electrophysiology to achieve the first direct measurement of the mitochondrial K+ and Cl- conductances. We named these conductances the mitochondrial uniporter for monovalent cations (UMC) and the mitochondrial voltage-gated Cl- channel (mClv). Direct electrophysiological analysis helped us identify their basic functional properties and eventually develop high- throughput assays to identify their inhibitors. Using these newly developed pharmacological tools, we demonstrated that UMC boosts mitochondrial capacity for oxidative phosphorylation, and its inhibition leads to an approximately five-fold increase in ATP production. In contrast, mClv supports mitochondrial anionic homeostasis and mitochondrial integrity by extruding excessive anionic substrates and byproducts of the Krebs cycle to the cytosol. In a comparison of mitochondria to an internal combustion engine, mClv is its exhaust system. In this project, we propose to achieve comprehensive electrophysiological characterization of UMC and mClv to define their functional properties. This will be followed by a detailed investigation into their mechanisms of action in intact mitochondria employing a combination of classical methods of mitochondrial bioenergetics (respiration, swelling, and ΔΨ measurements), electron microscopy (including cryo-tomography), and mitochondrial metabolomics. Finally, we will explore various pathways for the molecular identification of UMC and mClv using genetic, omics, and biochemical approaches. Accomplishing these goals is expected to yield transformative discoveries in the field of mitochondrial bioenergetics, advancing our understanding of this organelle beyond the original postulates of the chemiosmotic theory.