Sequential interaction of chloride and proton ions with the fast gate steer the voltage-dependent gating in ClC-2 chloride channels

Abstract

The interaction of either H(+) or Cl(-) ions with the fast gate is the major source of voltage (V(m)) dependence in ClC Cl(-) channels. However, the mechanism by which these ions confer V(m) dependence to the ClC-2 Cl(-) channel remains unclear. By determining the V(m) dependence of normalized conductance (G(norm)(V(m))), an index of open probability, ClC-2 gating was studied at different [H(+)](i), [H(+)](o) and [Cl(-)](i). Changing [H(+)](i) by five orders of magnitude whilst [Cl(-)](i)/[Cl(-)](o) = 140/140 or 10/140 mm slightly shifted G(norm)(V(m)) to negative V(m) without altering the onset kinetics; however, channel closing was slower at acidic pH(i). A similar change in [H(+)](o) with [Cl(-)](i)/[Cl(-)](o) = 140/140 mm enhanced G(norm) in a bell-shaped manner and shifted G(norm)(V(m)) curves to positive V(m). Importantly, G(norm) was >0 with [H(+)](o) = 10(-10) m but channel closing was slower when [H(+)](o) or [Cl(-)](i) increased implying that ClC-2 was opened without protonation and that external H(+) and/or internal Cl(-) ions stabilized the open conformation. The analysis of kinetics and steady-state properties at different [H(+)](o) and [Cl(-)](i) was carried out using a gating Scheme coupled to Cl(-) permeation. Unlike previous results showing V(m)-dependent protonation, our analysis revealed that fast gate protonation was V(m) and Cl(-) independent and the equilibrium constant for closed–open transition of unprotonated channels was facilitated by elevated [Cl(-)](i) in a V(m)-dependent manner. Hence a V(m) dependence of pore occupancy by Cl(-) induces a conformational change in unprotonated closed channels, before the pore opens, and the open conformation is stabilized by Cl(-) occupancy and V(m)-independent protonation.

Figure 1. Regulation of I_Cl through ClC-2 by [H⁺]_i

A, representative I_Cl(t) recordings obtained from 6 different cells dialysed with internal solutions whose [H⁺] (in m) was adjusted to the indicated values. Currents were recorded from −160 to +40 mV, in 40 mV increments. Horizontal (200 ms) and vertical (2 nA) bars are valid for all records. [H⁺]_o = 10^−7.3 m and [Cl⁻]_i = [Cl⁻]_o = 140 mm. B, fast (open circles) and slow (open squares) time constants as a function of [H⁺]_i were obtained by fitting eqn (2) to the onset phase of I_Cl(t). C, time constants of closing as a function of [H⁺]_i were obtained by fitting eqn (3) to the offset phase of I_Cl(t) at +60 mV. D, total I_Cl at −120 mV as a function of [H⁺]_i. n = 5–11 different cells.

Figure 2. ClC-2 gating at different [H⁺]_i

A, G_norm(V_m) curves at different [H⁺]_i. B, P_P(V_m) curves at different [H⁺]_i. [H⁺]_i (in m) and number of experiments were: 10^−9.1, 9 (⊲); 10^−8.2, 5 (⋄); 10^−7.3, 11 (▿); 10^−6.4, 11 (▵); 10^−5.5, 11 (○); 10^−4.0, 7 (□). Continuous lines in A and B are fits with Boltzmann eqn (1). C, estimated V_1/2 and z values obtained by fitting with eqn (1) individual G_norm(V_m) or P_P(V_m) curves. Open symbols are parameters for G_norm(V_m) curves while filled symbols are parameters corresponding to P_P(V_m) curves. D, G_norm(V_m) curves obtained from cells bathed first in a solution with pH 7.3 (filled squares) and then switched to a solution with pH 6.4 (open squares). The [Cl⁻]_i and pH_i were 10 mm and 6.4, respectively. [Cl⁻]_o was 140 mm.

Figure 3. Effect of [H⁺]_o on ClC-2 gating

A, I_Cl(t) recordings obtained from 4 different cells exposed first to control solution containing [H⁺]_o = 10^−7.3 m (left traces) and then to a test solution (right traces) whose [H⁺]_o was adjusted to the indicated values (in m). Horizontal bars = 100 ms, valid for all recordings. Vertical bar = 2 nA is valid for control recordings and that at 10^−6.4 m H⁺; vertical bar = 0.5 nA is valid for all remaining recordings. pH_i 7.3 and [Cl⁻]_i = [Cl⁻]_o = 140 mm. Currents displayed were sampled at V_m: −160 to +40 mV in 20 mV increments for 10^−6.4, 10^−8.2 and 10^−9.1 m H⁺, and from −200 to +40 for 10⁻¹⁰ m H⁺ in 40 mV increments. B, G_norm(V_m) curves at different [H⁺]_o constructed using data like those shown in A. Continuous lines are fits with Boltzmann eqn (1) used to estimate V_1/2 and z values. n = 4 (10⁻¹⁰, ◆),5 (10^−9.1, ▸), 5 (10^−8.2, ◂), 11 (10^−7.3, ▾), and 6 (10^−6.4, ▴). C, V_1/2 values at different [H⁺]_o. D, z values at different [H⁺]_o.

Figure 4. External H⁺ ions hinder channel closing

A, time course of G_norm at −110 (channel opening) and +60 (channel closing) mV and at different [H⁺]_o (10^−10.2 m green; 10^−9.1 m grey; 10^−7.3 m red; 10^−6.4 m light blue). Each trace is the average of four independent records. Broken lines at the top of the G_norm at −110 mV and +60 mV are fits with a mono-exponential function used to calculate the activation time constant (τ) or the closing time constant (τ_C) at each [H⁺]_o. B, τvs.[H⁺]_o curve. C, τ_C vs.[H⁺]_o curve. For each pH_o 5 < n < 9. pH_i 7.3 and [Cl⁻]_i = [Cl⁻]_o = 140 mm.

Figure 5. V_m-dependent gating of ClC-2 at different [H⁺]_o and [Cl⁻]_i ions

A, B, C and D, families of G_norm(V_m) curves at different [H⁺]_o from cells dialysed with 40 (A, 5 < n < 9), 80 (B, 4 < n < 10), 140 (C, 4 < n < 11) and 200 (D, 4 < n < 8) mm Cl⁻. [H⁺]_o were: 10^−9.1 (▾), 10^−8.4 (▴), 10^−7.3 (•;), and 10^−6.4 m (▪). The [H⁺]_i = 10^−7.3 m and [Cl⁻]_o = 140 mm was the same in all cases. Note that G_norm was >0 when the [H⁺]_o was 10^−9.1 m in all cases. Continuous lines are fits with eqn (6) (from Scheme II) used to calculate pK_O and β_o/α₀ ratio. The resulting parameters were: [Cl⁻]_i = 40 mm: B = 2.95, A = 1492, z = −1.02, pK_O = 7.8, z_h = −0.017; [Cl⁻]_i = 80 mm: B = 5.59, A = 350, z = −0.89, pK_O = 7.9, z_h = −0.016; [Cl⁻]_i = 140 mm: B = 3.2, A = 255, z = −0.83, pK_O = 7.96, z_h = 0; [Cl⁻]_i = 200 mm: B = 3.1, A = 68.8, z = −0.71, pK_O = 8.0, z_h = 0.008. SEM were similar for all V_m and for clarity only high and low [H⁺]_o are included. E, pK_O vs. V_m at different [Cl⁻]_i. F, β₀/α₀ ratio vs. V_m curves for C↔O transition (Scheme II) at different [Cl⁻]_i. Continuous lines are fits using eqn (7) with z = −0.79, −0.75, −0.75 and −1.13 for 40, 80, 140 and 200 mm Cl⁻, respectively. G, τ_C vs.[H⁺]_o at different [Cl⁻]_i. Continuous lines are fits with eqn (9). For E, F and G curves [Cl⁻]_i was (in mm): (□ = 40; ○ = 80; ▵ = 140, and ▿ = 200 mm). Triangles and inverted triangles plotted at +60 mV in panel E are pK_o values obtained from fitting eqn (9) to data shown in panel G.

Figure 6. Model 2 for ClC-2 gating

A, kinetic model built according to Scheme II that couples movement of the fast gate to Cl⁻ occupancy of the pore_. Rectangular boxes indicate four conformations: C = closed unprotonated, O = open unprotonated, C_H = closed protonated and O_H = open protonated. S_cen and S_ext anion binding sites are represented by dashed green and red circles, respectively. Cl⁻ is represented by a green sphere, H⁺ by a blue sphere and the fast gate by a red sphere. Protonation of the fast gate by is indicated by a blue sphere attach to a red sphere. Transitions between Cl⁻-free and Cl⁻-bound states are dictated by V_m-dependent rate constants. Transitions between unprotonated and protonated states are characterized by V_m-independent equilibrium constants and K_O, respectively. Fast gate is open by electrostatic repulsion when either a Cl⁻ (f₁ = 151) or H⁺ ion is bound (f_H = 8.46). B, simulated ionic currents from +60 mV to −180 mV, in 40 mV increments according to the voltage protocol shown on top. I_Cl(t) were simulated for [Cl⁻]_i = [Cl⁻]_o = 140 mm, pH_o = pH_i = 7.3. C, simulated time course of G_norm at −110 and +60 with [H⁺]_o from = 10⁻¹⁰ m to = 10^−6.4 m. D, opening time constant vs.[H⁺]_o. E, closing time constant vs.[H⁺]_o. Simulations were done using the parameter set listed in Table 1.

Figure 7. Simulation of steady-state properties of ClC-2 gating under different ionic conditions according to Model 2

A, G_norm(V_m) dependence on [H⁺]_o (from 10⁻¹⁰ to 10^−6.4 m) was simulated using Model 2. B, simulations of the dependence on [Cl⁻]_i (from 10 to 200 mm) of the G_norm(V_m) curve. C, lack of effect of [Cl⁻]_o on G_norm(V_m). Curves were simulated using [Cl⁻]_o ranging between 10 and 200 mm. D, energy barriers experienced by a Cl⁻ ion going through the permeation pathway in ClC-2 at the indicated V_m. Energy profiles were calculated considering that permeation and gating are coupled as described by Model 2. E, voltage dependence of probability of ClC-2 pore occupancy by Cl⁻ at the indicated [Cl⁻]_i. F, voltage dependence of probability of ClC-2 pore occupancy by Cl⁻ is independent of [Cl⁻]_o. All simulations were done using the parameter set listed in Table 1.