Permeant anions contribute to voltage dependence of ClC-2 chloride channel by interacting with the protopore gate

Abstract

It has been shown that the voltage (V(m)) dependence of ClC Cl(-) channels is conferred by interaction of the protopore gate with H(+) ions. However, in this paper we present evidence which indicates that permeant Cl(-) ions contribute to V(m)-dependent gating of the broadly distributed ClC-2 Cl() channel. The apparent open probability (P(A)) of ClC-2 was enhanced either by changing the [Cl(-)](i) from 10 to 200 mM or by keeping the [Cl(-)](i) low (10 mM) and then raising [Cl(-)](o) from 10 to 140 mM. Additionally, these changes in [Cl(-)] slowed down channel closing at positive V(m) suggesting that high [Cl(-)] increased pore occupancy thus hindering closing of the protopore gate. The identity of the permeant anion was also important since the P(A)(V(m)) curves were nearly identical with Cl(-) or Br(-) but shifted to negative voltages in the presence of SCN() ions. In addition, gating, closing rate and reversal potential displayed anomalous mole fraction behaviour in a SCN(-)/Cl() mixture in agreement with the idea that pore occupancy by different permeant anions modifies the V(m) dependence ClC-2 gating. Based on the ec1-ClC anion pathway, we hypothesized that opening of the protopore gate is facilitated when Cl(-) ions dwell in the central binding site. In contrast, when Cl(-) ions dwell in the external binding site they prevent the gate from closing. Finally, this Cl(-)-dependent gating in ClC-2 channels is of physiological relevance since an increase in [Cl(-)](o) enhances channel opening when the [Cl(-)](i) is in the physiological range.

Figure 2. [Cl⁻]_o dependence of ClC-2 gating

A, I_Cl(t) at different V_m values (+40 to −160 mV) was recorded from the same cell dialysed with 140 mm Cl⁻ and bathed in solutions containing 40, 80 and 140 mm [Cl⁻]_o. B, I_Cl(t) at different V_m values recorded from the same cell dialysed with 10 mm Cl⁻ and consecutively bathed in solutions containing 10, 40 and 140 mm [Cl⁻]_o. C, P_A(V_m) curves for cells dialysed with 140 mm (open symbols) or 10 mm (filled symbols) Cl⁻ and bathed in solutions containing [Cl⁻]_o = 10 , 40 (▸, ▵), 80 (▿), 140 (•, ▪) and 200 (⋄) mm. The number of independent cells (n) tested were 5, 5, and 6, for [Cl⁻]_i = 140 and [Cl⁻]_o = 40, 80 and 200 mm, respectively and 6, 5, and 6, for [Cl⁻]_i = 10 and [Cl⁻]_o = 10, 40 and 140 mm, respectively. Continuous lines through P_A(V_m) values are fits with the Boltzmann equation. D, V_1/2 values are plotted against the [Cl⁻]_o. E, z against [Cl⁻]_o. Open and filled symbols in D and E are data obtained with 140 and 10 mm intracellular Cl⁻, respectively. In cells dialysed with 10 mm [Cl⁻], expected E_Cl and measured V_r values were (in mV): 0 and −0.1 ± 0.9; −35.5 and −33.8 ± 5.3; −67.5 and −70.1 ± 2.3.

Figure 1. [Cl⁻]_i dependence of ClC-2 gating

A, I_Cl(t) recorded from five different cells dialysed with 10, 40, 80, 140 and 200 mm [Cl⁻]_i. [Cl⁻]_o = 140 mm and pH_i = pH_o = 7.3. Horizontal bars (200 ms, except for 10 and 40 mm, where it is 50 ms) and vertical bars (1 nA, except for 200 mm, where it is 2 nA) displayed are valid for all traces. V_m varied from −200 to +40 mV, in 40 mV increments. B, P_A(V_m) curves constructed at different [Cl⁻]_i. [Cl⁻]_i (in mm) and number of experiments (n) were: 10, 5 (□); 40, 9 (▵); 80, 10 (▿); 140, 11 (•); and 200, 7 (⋄). Continuous black lines through data are Boltzmann fits used to determine V_0.5 and z values. C, P_P(V_m) curves constructed at different [Cl⁻]_i. The P_P was determined by using a double pulse protocol (see Methods for details). [Cl⁻]_i (in mm) and n values were the same as in B. The continuous lines are Boltzmann fits used to determine V_1/2 and z values. D, V_1/2 values for P_A(V_m) and P_P(V_m) are plotted against the [Cl⁻]_i as filled and open circles, respectively. E, z values at different [Cl⁻]_i for data shown in B (filled circles) and C (open circles). Expected E_Cl and measured V_r values were (in mV): −67.5 and −70.1 ± 2.4; −32 and −32.7 ± 1.1; −14.3 and −14.4 ± 2.1; 0 and 0.1 ± 1.4; 9.1 and 9.8 ± 0.23.

Figure 3. Gating of ClC-2 in the presence of Cl⁻, Br⁻ and SCN⁻

A, representative ClC-2 currents recorded from three different cells bathed and dialysed with solutions containing 140 mm Cl⁻ (left), 140 mm Br⁻ (centre) or 140 mm SCN⁻ (right). B, V_m dependence of τ_f, τ_s, A_f and A_s describing the onset of the ClC-2 currents recorded using solutions containing Cl⁻ (•), Br⁻ (▪) or SCN⁻ (□). C, G_norm(t) from cells bathed and dialysed with solutions containing 140 mm Cl⁻, 140 mm Br⁻ or 140 mm SCN⁻. Each trace is the average of 5, 3 and 5 different cells voltage clamped at −140 and then repolarized to +60 mV. D, τ_c values calculated by fitting a mono-exponential function to G_norm at +60 mV are shown for each ionic condition. E, V_m dependence of P_A in the presence of Cl⁻ (•), of Br⁻ (▪) and of SCN⁻ (□). Continuous lines are Boltzmann fits used to calculate V_1/2 and z parameters. P_A was calculated from instantaneous I_tail recorded at +60 mV. The number of experiments (n) was 5, 4 and 5 for Cl⁻, Br⁻ and SCN⁻, respectively.

Figure 4. Effect of extracellular SCN⁻ mole fractions on ClC-2 gating

A, I_Cl/SCN(t) at the indicated extracellular SCN⁻ mole fractions. [Cl⁻]_i = 140 mm, pH_o = pH_i = 7.3. V_m varied from −160 to +40 mV, in 40 mV increments. B, P_A(V_m) curves from cells dialysed with different extracellular SCN⁻ mole fractions (• = 0, ▴ = 0.25; ▾ = 0.5; ♦ = 0.75 and ◂ = 1). C, D and E, effect of external SCN⁻ mole fractions on V_r, V_1/2 and z, respectively. n = 5–10.

Figure 5. Anomalous mole fraction behaviour of ClC-2 gating induced by intracellular SCN⁻ mole fractions

A, I_Cl/SCN(t) at indicated intracellular SCN⁻ mole fractions. [Cl⁻]_o = 140 mm, pH_o = pH_i = 7.3. V_m varied from −160 to +40 mV, in 40 mV increments. B, P_A(V_m) curves from cells dialysed with SCN⁻ mole fractions equal to 0 (○), 0.05 (□), 0.25 (▵), 0.5 (▿), 0.75 (⋄) and 1 (◃). C, D and E, anomalous mole fraction behaviour of V_r, V_1/2 and z, respectively. n = 5–11.

Figure 6. Permeant anions interact with the protopore gate to tune ClC-2 gating

Upper panel: time course of G_norm calculated from whole cell currents activated by square voltage pulses from 0 to −140 (grey bars) and then to +60 mV (black bars). Lower panel: corresponding τ_c at +60 mV calculated from data like those shown in the upper panel by fitting G_norm at +60 mV with eqn (3) (grey lines). Closing rate was slowed down by increasing [Cl⁻]_i (A) or [Cl⁻]_o (B), displayed anomalous mole fraction behaviour with intracellular SCN⁻ mole fraction (C) and was accelerated by increasing external SCN⁻ mole fractions (D). n = 5–10 for each ionic condition.

Figure 7. External Cl⁻ ions can interact with the protopore gate to modify the ClC-2 gating when [Cl⁻]_i was maintained at 10 mm

A, time course of G_norm calculated from whole cell currents activated by a square voltage pulse from 0 to −140 (grey bars) and then to +80 mV (black bars). Traces obtained with 10 and 140 mm [Cl⁻]_o are indicated by arrows. B, corresponding τ_c at +80 mV. n = 5.

Scheme 1