Oscar Teijido Hermida

A Putative Role of Voltage-Dependent Anion Channel in Ischemia

Ischemia is characterized by the inhibition of ATP production via oxidative phosphorylation due to oxygen deprivation, which leads to mitochondria depolarization. Impaired mitochondrial respiration involves the shift to an anaerobic metabolism and ionic imbalance that leads to a decrease of cytosolic pH and generation of reactive oxygen species (ROS). The voltage-dependent anion channel (VDAC) is the most abundant protein in the mitochondrial outer membrane, which regulates the passage of ATP, ADP, and other mitochondrial respiratory substrates across this membrane. Motivated by these facts, we are interested in establishing a possible functional link between the physiological features of ischemia and VDAC. It has been reported that cytosolic acidification protects cells from death during early ischemic stress. However, the mechanism of this protection is still unclear. Ischemia is followed by a reperfusion stage in which pH is restored to neutral leading to cell death. In experiments with VDAC reconstituted into planar lipid membranes we show that VDAC voltage-induced closure increases at low pH. Interestingly, the channel fully re-opens after the pH is restored to neutral. The cytoskeleton protein, dimeric tubulin regulates mitochondrial membrane potential by blocking VDAC permeability for ATP (Sheldon et al., 2011; Gurnev et. al, 2011). We found that, synergistically with the effect on gating, acidification significantly promotes VDAC blockage by tubulin, and speculate here that acidic pH-induced VDAC inhibition could potentially be involved in the protective effect of acidosis and also in the mitochondria depolarization usually detected during ischemia.A possible effect of ROS on VDAC permeability was assessed by probing VDAC functioning in planar membranes containing oxidized lipids. We have found that oxidized lipids affect VDAC interaction with tubulin. The relevance of these observations for the mechanisms of ischemia and apoptosis is discussed.

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

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.