Current Insights into the Molecular Mechanisms of VDAC-Tubulin Interaction

Abstract

Voltage-dependent anion channel (VDAC), the major channel of the mitochondrial outer membrane, serves as a principal pathway for ATP, ADP, and other respiratory substrates across this membrane. Recently, we found that free dimeric tubulin induces highly efficient reversible blockage of VDAC reconstituted into planar lipid membranes. Although the tubulin-blocked state is still conductive for small ions, it has reduced dimensions compared with open state, reversed ionic selectivity, and most importantly is virtually impermeable for ATP. We proposed that by blocking VDAC permeability, tubulin controls mitochondrial respiration. We suggested a model of VDAC-tubulin interaction where the negatively charged C-terminal tail (CTT) of tubulin partially blocks VDAC pore. The corresponding effective “gating charge” of the blockage is impressively high of about 10-14 elementary charges, which compares well with the gating charge of the most voltage-sensitive channels. Surprisingly, this gating charge only mildly depends on salt concentration and significantly exceeds the total number of negative charges of tubulin CTT. This indicates the applied field interacts not only with CTT but also with VDAC charges and dipoles, with all of them contributing to the effective gating charge. Our results also demonstrate that additional steps are involved in the blockage process. We found that lipid-dependent tubulin binding to the membrane greatly impacts VDAC blockage by tubulin. At 100 mM KCl, the charged lipids significantly affect the conductance of the tubulin-blocked state and the on-rate the blockage, but even at 1 M KCl the lipids with different non-lamellar tendencies were shown to change the on-rate by two orders of magnitude. These data allowed us to conclude that hydrophobic interactions between the tubulin and the membrane represent an essential component of the multistep process of VDAC blockage by tubulin, providing a new instructive example of lipid-controlled protein-protein interactions.