Investigating VDAC Gating via Magic Angle Spinning NMR and Electrophysiological Measurements Under Extreme pH Conditions: Implications for the Voltage-Gating Mechanism

Abstract

The voltage-dependent anion-selective channel (VDAC) is an integral membrane protein that controls transportation of metabolites across the outer mitochondrial membrane. The mechanism by which VDAC controls ion flow is important to understanding cellular metabolic processes. It remains an unresolved problem despite publication of the structure of VDAC's open conformation. According to the first gating model proposed by Marco Colombini's group, gating occurs concurrently with large structural rearrangements, and there exist multiple conformations of closed states. These closed states have not been examined by diffraction or NMR, and thus obtaining structural information on VDAC under conditions that promote closure are valuable to unraveling the gating mechanism.Accordingly we have used magic angle spinning NMR (MAS NMR) and electrophysiological measurements to study recombinant human VDAC1 in lipid bilayers under extreme pH conditions that have been shown to perturb VDAC gating and structure. Detergent-solubilzed membrane proteins are often not amenable to solution NMR studies at extreme pH conditions; thus MAS NMR is uniquely positioned to study the structure and dynamics of closed conformations of VDAC in lipid bilayers. At pH 4 and lower we observe changes in chemical shifts and peak intensities for some, but not all, residues in VDAC's N-terminus, and for residues not in the voltage-sensing domain. Changes in chemical shifts for some residues in the N-terminus are reversed when the pH is raised from low to neutral values. Electrophysiological experiments with VDAC reconstituted into a planar lipid membrane confirmed that VDAC functions properly at pH values as low as 3.0. Furthermore, low pH enhances voltage-gating, and this pH effect is fully reversible. We discuss the implications of our observations and evaluate possible gating mechanisms.