Affixing N-terminal α-Helix to the Wall of the Voltage-dependent Anion Channel Does Not Prevent Its Voltage Gating

Abstract

Background: There is ongoing controversy concerning the location and mobility of the N-terminal ␣-helix in VDAC1 during voltage gating. Results: mVDAC1 with the N-terminal ␣-helix cross-linked to ␤-strand 11 forms typical voltage-gated channels. Conclusion:The N-terminal domain of VDAC1 does not move independently during voltage gating. Significance: This study dramatically alters the current view of voltage gating dynamic in VDAC1.The voltage-dependent anion channel (VDAC) governs the free exchange of ions and metabolites between the mitochondria and the rest of the cell. The three-dimensional structure of VDAC1 reveals a channel formed by 19 ␤-strands and an N-terminal ␣-helix located near the midpoint of the pore. The position of this ␣-helix causes a narrowing of the cavity, but ample space for metabolite passage remains. The participation of the N-terminus of VDAC1 in the voltage-gating process has been well established, but the molecular mechanism continues to be debated; however, the majority of models entail large conformational changes of this N-terminal segment. Here we report that the pore-lining N-terminal ␣-helix does not undergo independent structural rearrangements during channel gating. We engineered a double Cys mutant in murine VDAC1 that cross-links the ␣-helix to the wall of the ␤-barrel pore and reconstituted the modified protein into planar lipid bilayers. The modified murine VDAC1 exhibited typical voltage gating. These results suggest that the N-terminal ␣-helix is located inside the pore of VDAC in the open state and remains associated with ␤-strand 11 of the pore wall during voltage gating.The voltage-dependent anion channel (VDAC) 4 serves as the primary conduit between the mitochondria and the rest of the cell, facilitating free exchange of ions and metabolites across the outer mitochondrial membrane. In addition to its metabolic and energetic functions, VDAC has a more complex role, serving as a receptor for molecules and proteins that modulate the organelle's permeability and thereby its function (1-3). This multifunctional channel has been implicated in the metabolic stresses of cancer, cardiovascular disease, and mitochondrialdependent cell death (4 -8); thus, understanding the structure and function of VDAC constitutes a critical objective for basic as well as medical research.Single channel conductance experiments on VDAC1 at low membrane potential (Ͻ30 mV) reveal a high conductance (4.1 Ϯ 0.1 nanosiemens in 1 M KCl) indicative of a large pore, usually referred to as the "open" state of the channel (1). This conformer facilitates the passage of 10 6 ATP molecules (9) and displays a preference for monovalent anions over cations with the anion-to-cation permeability ratio of 2:1 in high salt (10) and 4:1 in physiological salt concentration (11). As voltage is increased (Ͼ30 mV) in either a positive or a negative direction, the channel switches into the lower conducting states (around 2 nanosiemens in 1 M KCl), termed as the "closed" state(s). These states are cation-selective wi...

High Resolution Structure and Double Electron-Electron Resonance of the Zebrafish Voltage-dependent Anion Channel 2 Reveal an Oligomeric Population

“…There have now been a number of studies confirming that this conformation probably resides in the "open" state of the channel (11,12).…”

“…Numerous studies have established the functional importance of the N-terminal ␣-helix in regulating the flux of metabolites through the channel (9,10), and the observation that it is localized within the pore makes it ideally suited to regulate the passage of ions and metabolites through the pore. Based on molecular dynamics simulations and a series of cross-linking studies, the structure is believed to reside in the "open" conformation, facilitating the passage of ATP and other nucleotides (11,12).…”

Voltage-dependent structural changes of the membrane-bound anion channel hVDAC1 probed by SEIRA and electrochemical impedance spectroscopy

“…17,18 Recent studies focused on the role of the a-helix by fixing it covalently to the barrel wall. 18,19 Mertins et al 18 demonstrated that fixing the helix at two different positions either impaired voltage-induced closure of the channel or produced an asymmetric gating that depended on the direction of the electric field. In contrast, Teijido et al 19 found voltageinduced gating despite restricted helix mobility.…”

Nucleotide Interactions of the Human Voltage-dependent Anion Channel

“…Electron microscopy and bilayer measurements suggest moderate to large structural changes during channel closure (5,9,20). Models for gating mechanisms include rearrangements of the N-terminal ␣-helix, barrel deformations, a combination of helix and barrel motions to varying extent, and translocation of a membrane-embedded voltage sensor out of the membrane (4,(21)(22)(23)(24)(25)(26)(27). Although NADH enhances the channel voltage dependence and reduces nucleotide permeability of the outer mitochondrial membrane, ATP itself has no effect on VDAC gating (16,28).…”