Concentration dependent ion selectivity in VDAC: a molecular dynamics simulation study

Abstract

The voltage-dependent anion channel (VDAC) forms the major pore in the outer mitochondrial membrane. Its high conducting open state features a moderate anion selectivity. There is some evidence indicating that the electrophysiological properties of VDAC vary with the salt concentration. Using a theoretical approach the molecular basis for this concentration dependence was investigated. Molecular dynamics simulations and continuum electrostatic calculations performed on the mouse VDAC1 isoform clearly demonstrate that the distribution of fixed charges in the channel creates an electric field, which determines the anion preference of VDAC at low salt concentration. Increasing the salt concentration in the bulk results in a higher concentration of ions in the VDAC wide pore. This event induces a large electrostatic screening of the charged residues promoting a less anion selective channel. Residues that are responsible for the electrostatic pattern of the channel were identified using the molecular dynamics trajectories. Some of these residues are found to be conserved suggesting that ion permeation between different VDAC species occurs through a common mechanism. This inference is buttressed by electrophysiological experiments performed on bean VDAC32 protein akin to mouse VDAC.

Figure 1. The structure of VDAC.

Top and side view of the three atomic resolution structures determined for (A) mVDAC1 and (B,C) hVDAC1 determined by either Xray crystallography (A; pdb code 3emn), by NMR (B; pdb code 2k4t), or by a combination of both methods (C; pdb code 2jk4) –. These structures mainly differ in the position of the N-terminal segment (highlighted in red). The protein structures are depicted as cartoon. Only the best representative conformer is shown for the NMR structure. All images were prepared with vmd .

Figure 2. Translocation of ions through VDAC.

The translocation path of (A) a Cl⁻ (in green) and (B) a K⁺ (in purple) ion through mVDAC1 was extracted from the first 0.1 M KCl simulation. The start and end positions of the permeating Cl⁻/K⁺ ion are highlighted as a green/purple sphere. The broken lines connect the positions of the chloride or potassium ion separated by a timestep of 4 ps. The protein is shown as a transparent-white cartoon.

Figure 3. The ion distribution inside mVDAC1 depends on the bulk KCl concentration.

(A) Number of Cl⁻ (green) and K⁺ (purple) ions inside the channel (N_pore) versus the bulk concentration ([KCl]_bulk). (B) Partition of Cl⁻ (green) and K⁺ (purple) ions in the pore relative to the bulk (N_pore/[KCl]_bulk) as a function of the bulk salt concentration [KCl]_bulk. (C) N_Cl−/N_K+ ratio inside the pore as a function of [KCl]_bulk. The data were extracted from two independent 15 ns simulations shown as crosses and stars, respectively at 0.2, 0.4, 0.6, 0.8, 1.0, 1.2 and 1.4 M KCl solution concentration and from two independent 50 ns simulations shown as circles at 0.1 and 1.0 M KCl solution concentration.

Figure 4. Ion pathways followed by K⁺ and Cl⁻ through mVDAC1 channel.

Positions of the K⁺ (purple; A, C) and Cl⁻ (green; B, D) ions within 10 Å of the protein residues are superimposed using 2000 snapshots extracted every 500 ps from the 100 ns trajectories at 0.1 M (A, B) and 1.0 M (C, D). The protein is shown as a transparent-white cartoon.

Figure 5. The electric field in mVDAC1 pore.

(A) Cross section along the z axis through the channel. The orientation of the time-averaged electric field computed using the first 0.1 M KCl trajectory is depicted by arrows. Its magnitude is given as a color scale shown on the right hand side. The electric field was calculated using the PMEPot module of vmd and is shown using OpenDX (http:/www.opendx.org). The field shows that at the two entrances of the pore chloride ions are attracted and cations are repelled. (B) Representation of the orientation of mVDAC1 as in the depicted cross section. A molecular-surface rendering and a ribbon representation are used for regions of the lipids and of the protein, respectively, located behind the plane of the cross section.

Figure 6. The ion translocation profile through the pore is concentration dependent.

(A) The electrostatic free energy profile computed using the PB approach (B) The free energy profile computed using the MD simulations. PB and MD permeation profiles were determined along the axis of the pore for K⁺ (purple) and Cl⁻ (green), in a KCl concentration of 0.1 M (solid line) and 1.0 M (dashed line).

Figure 7. Effect of ionic strength on the bean VDAC32 reversal potential.

The reversal potential (in open circles) is plotted against the salt concentration of the trans compartment. The concentration ratio was kept constant at a value of 2.0. The reversal potential calculated using the voltage GHK equation (with the P_Cl/P_K = 1.10 at a concentration ratio 2/1 M KCl (trans/cis)) is shown as a solid line. The reversal potential calculated using the Planck equation (diffusion coefficient of ion in solution D_Cl/D_K = 1.04) is shown as a dashed line.