Acidification asymmetrically affects voltage-dependent anion channel implicating the involvement of salt bridges

The voltage-dependent anion channel (VDAC) is the major pathway for ATP, ADP, and other respiratory substrates through the mitochondrial outer membrane, constituting a crucial point of mitochondrial metabolism regulation. VDAC is characterized by its ability to "gate" between an open and several "closed" states under applied voltage. In the early stages of tumorigenesis or during ischemia, partial or total absence of oxygen supply to cells results in cytosolic acidification. Motivated by these facts, we investigated the effects of pH variations on VDAC gating properties. We reconstituted VDAC into planar lipid membranes and found that acidification reversibly increases its voltage-dependent gating. Furthermore, both VDAC anion selectivity and single channel conductance increased with acidification, in agreement with the titration of the negatively charged VDAC residues at low pH values. Analysis of the pH dependences of the gating and open channel parameters yielded similar pKa values close to 4.0. We also found that the response of VDAC gating to acidification was highly asymmetric. The presumably cytosolic (cis) side of the channel was the most sensitive to acidification, whereas the mitochondrial intermembrane space (trans) side barely responded to pH changes. Molecular dynamic simulations suggested that stable salt bridges at the cis side, which are susceptible to disruption upon acidification, contribute to this asymmetry. The pronounced sensitivity of the cis side to pH variations found here in vitro might provide helpful insights into the regulatory role of VDAC in the protective effect of cytosolic acidification during ischemia in vivo.