Probing VDAC Voltage-Gating Mechanism by pH: Functional and Structural Implications

Abstract

VDAC controls fluxes of ATP/ADP and other respiratory substrates across mitochondrial outer membrane by using its characteristic ability to switch or “gate” between the so-called “open” and “closed” states. While most metabolites go freely through a unique open state, the closed states are virtually impermeable to ATP and ADP. Therefore, unveiling molecular mechanisms of VDAC gating is important in our understanding of mitochondrial respiration and metabolism in health and pathology. Available crystal structure of VDAC solved to the atomic level of resolution does not provide data on VDAC gating mechanism in spite of a number of different models that have been proposed. Although effects of pH on VDAC gating have been shown previously, here we further explore this approach by performing functional and structural studies on VDAC at extremely low pH. In our experiments with VDAC reconstituted into planar lipid membranes, voltage-gating is drastically increased as pH decreases from 7.4 to 3.0. Interestingly, the effect of pH on gating is fully reversible, i.e., gating returns to the initial behavior after returning pH back to 7.4. Further, we explore the effects of pH on channel selectivity and protein-protein interaction of VDAC with dimeric tubulin that is known to block VDAC pore with nanomolar efficiency. To address structural rearrangements upon gating, we also use magic angle spinning NMR to study conformational changes in recombinant human VDAC1 as a function of pH ranging from 3 to 11. Under these conditions we observe reversible changes in chemical shifts upon changing the pH from 7 to 4. These observations support functional results of VDAC voltage-gating at different pH and complement the data with site-specific information about residues affected by pH changes. The mechanism of VDAC gating and its relevance to in vivo situation are discussed.