Defining the roles and regulation of the mitochondrial VDAC isoforms one molecule at a time

The voltage-dependent anion channel (VDAC) is recognized as the key metabolite conduit in the mitochondrial outer membrane and regulator for fluxes of water-soluble metabolites, ions, and polypeptides. The uniqueness of VDAC arises from its position at the interface between the mitochondria and the cytosol making VDAC an important cellular communication hub. VDAC has three isoforms in mammals: VDAC1, 2, and 3 which share high sequence similarity, indicative of a similar pore-forming structure. All three VDAC isoforms form similar highly conductive, anion-selective, voltage-gated channels when reconstituted into planar lipid membranes implying that they can concurrently facilitate the transport of the negatively charged respiratory metabolites. However, cellular model systems and isoform-specific knock-out mice demonstrate distinctive functional roles for each VDAC isoform. We explored the biophysical basis for these differences by reconstituting human, mouse, and zebrafish VDAC isoforms in planar lipid bilayers and performing single channel electrophysiology. We have uncovered that interactions with cytosolic regulators, such as the Parkinson’s disease associated alpha-synuclein, vary in their on-rates by two-orders of magnitude between each isoform. Furthermore, each isoform varies in their ability translocate synuclein across the lipid bilayer. Exploring the calcium transport properties of each isoform, we show that VDAC3 has the highest calcium permeability. We demonstrate that VDAC2 can dynamically switch between channel properties of VDAC1 and VDAC3, and explored the structural basis for its unique plasticity using mutational and structural analysis and molecular dynamics simulations. We find that both VDAC2’s N-terminal extension and cysteine residues contribute to, but are not sufficient to explain VDAC2’s striking behavior. Our results suggest that tuning of beta-barrel and loop flexibility between the isoforms could define their biophysical properties and help to explain distinct physiological roles.