Abstract
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.
Highlights
A representative experiment illustrated by Fig. 1 demonstrates a dramatic difference in voltage-dependent anion channel (VDAC) behavior between pH 7.4 and 5.5, with the pH symmetrically modified on both, cis and trans, sides of the membrane
At pH 7.4, VDAC remains in a high conducting open state of G ϭ 4.1 Ϯ 0.1 nanosiemens at both Ϯ 20 and Ϯ 30 mV (Fig. 1, A and B, upper traces)
It has been demonstrated in a number of studies [3, 23, 35, 36] that different charged residues could be responsible for the channel gating, conductance, and ion selectivity
Summary
We reconstituted VDAC into planar lipid membranes and found that acidification reversibly increases its voltage-dependent gating 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. We found that the number of salt bridges that can potentially be disrupted at low pH is significantly higher in the cytosolic side compared with the mitochondrial side This finding could help to explain the asymmetric response of channel gating to acidification. The asymmetric sensitivity of VDAC to acidification in favor of the cytosolic side of the channel and the full reversibility of this pH response may provide new insights into this “pH paradox” of ischemia/reperfusion injury
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