The fast gating mechanism in ClC-0 channels

Abstract

We investigate and then modify the hypothesis that a glutamate side chain acts as the fast gate in ClC-0 channels. We first create a putative open-state configuration of the prokaryotic ClC Cl- channel using its crystallographic structure as a basis. Then, retaining the same pore shape, the prokaryotic ClC channel is converted to ClC-0 by replacing all the nonconserved polar and charged residues. Using this open-state channel model, we carry out molecular dynamics simulations to study how the glutamate side chain can move between open and closed configurations. When the side chain extends toward the extracellular end of the channel, it presents an electrostatic barrier to Cl- conduction. However, external Cl- ions can push the side chain into a more central position where, pressed against the channel wall, it does not impede the motion of Cl- ions. Additionally, a proton from a low-pH external solution can neutralize the extended glutamate side chain, which also removes the barrier to conduction. Finally, we use Brownian dynamics simulations to demonstrate the influence of membrane potential and external Cl- concentration on channel open probability.

FIGURE 1

(A) The MD system with the ClC-0 monomer cross sectioned along the pore and represented by a solvent accessible surface. The water column extends to 90 Å total height. Glu-148 is highlighted at the center of the pore, with the two negatively charged oxygens of its side chain in red. (B) Detail view of the central region. (C) The same region in a model with a smaller minimum pore diameter (≈4 Å). Two Cl⁻ ions are shown in green, radius 1.81 Å.

FIGURE 2

Positions of Cl⁻ ions and the Glu-148 side chain during conduction attempts. Cl⁻ ions are shown in green, Glu-148 in yellow with side-chain oxygens red; all atoms and ions are displayed at van der Waals radii. (A) Inward conduction: the lower Cl⁻ ion (Cl-3) travels down past the Glu-148 side chain in its open configuration, while another ion (Cl-4) remains on the extracellular side of the gate. (B) During attempted outward conduction, the Glu-148 side chain has swung upward, blocking the pore. Ions Cl-2 (lower) and Cl-3 (upper) are shown. (C) For successful outward conduction, a third Cl⁻ ion (Cl-3, uppermost) keeps the side chain in the open position while the middle Cl⁻ ion (Cl-2) moves upward, influenced by Cl-1 (lowest). Cl-2 is about to replace Cl-3 (the “knock-on” effect).

FIGURE 3

Energy profiles for a Cl⁻ ion passing the channel gate. (Lower lines) The energy of the system is calculated as the ion moves through the pore with the Glu-148 side chain in the open position at z ≈ 1 Å (see Fig. 2 A) and a second Cl⁻ ion fixed at z = −8 Å. (Upper lines) The side chain is in the closed configuration, stretched upward to z ≈ 5 Å (see Fig. 2 B), and a second Cl⁻ ion is fixed at z = −6 Å. Dielectric constants of 35 (solid lines) and 60 (dashed lines) are used in the pore.

FIGURE 4

Fast gate opening to allow inward conduction. The upper curve shows the z position of Cl-4, and the lower curve shows the distance between this ion and Cl-3. Cl-4 is constrained near specified positions during the simulation, while the other ions and the Glu-148 side chain are free. Initial positions of ions and the side chain are sketched in the first inset. When Cl-4 is brought into the pore, the resulting configuration (middle inset) is relatively stable for ∼300 ps. But the side chain eventually retreats against the wall of the pore for a moment and Cl-3 slips past (right-hand inset).

FIGURE 5

Energy profiles for a Cl⁻ ion when the Glu-148 side chain is extended toward the external vestibule, assuming dielectric constant of 35 for water in the pore. (Solid line) Protonated side chain, with a second Cl⁻ ion at z = −3 Å. (Dashed line) unprotonated side chain, with a second ion at z = −6 Å (reproduced from Fig. 3).

FIGURE 6

Mean approach times from Brownian dynamics simulations. The mean time for a Cl⁻ ion to enter the outer vestibule and approach within 7 Å from the Glu-148 side chain (closed configuration) is plotted against membrane potential. External Cl⁻ concentration is 150 mM (•) or 50 mM (○). Each data point represents the average of 32–48 separate runs. Error bars have a length of 1 ± SE and are not shown when they are smaller than the data points.