Proton-dependent inhibition, inverted voltage activation, and slow gating of CLC-0 Chloride Channel

Abstract

CLC-0, a prototype Cl- channel in the CLC family, employs two gating mechanisms that control its ion-permeation pore: fast gating and slow gating. The negatively-charged sidechain of a pore glutamate residue, E166, is known to be the fast gate, and the swinging of this sidechain opens or closes the pore of CLC-0 on the millisecond time scale. The other gating mechanism, slow gating, operates with much slower kinetics in the range of seconds to tens or even hundreds of seconds, and it is thought to involve still-unknown conformational rearrangements. Here, we find that low intracellular pH (pHi) facilitates the closure of the CLC-0's slow gate, thus generating current inhibition. The rate of low pHi-induced current inhibition increases with intracellular H+ concentration ([H+]i)-the time constants of current inhibition by low pHi = 4.5, 5.5 and 6 are roughly 0.1, 1 and 10 sec, respectively, at room temperature. In comparison, the time constant of the slow gate closure at pHi = 7.4 at room temperature is hundreds of seconds. The inhibition by low pHi is significantly less prominent in mutants favoring the slow-gate open state (such as C212S and Y512A), further supporting the fact that intracellular H+ enhances the slow-gate closure in CLC-0. A fast inhibition by low pHi causes an apparent inverted voltage-dependent activation in the wild-type CLC-0, a behavior similar to those in some channel mutants such as V490W in which only membrane hyperpolarization can open the channel. Interestingly, when V490W mutation is constructed in the background of C212S or Y512A mutation, the inverted voltage-dependent activation disappears. We propose that the slow kinetics of CLC-0's slow-gate closure may be due to low [H+]i rather than due to the proposed large conformational change of the channel protein. Our results also suggest that the inverted voltage-dependent opening observed in some mutant channels may result from fast closure of the slow gate by the mutations.

Fig 1. Structure of vertebrate CLC channels.

Structure of human CLC-1 molecule (PDB accessing code: 6QVU) is used to represent the structure of CLC-0, which is still not available. CLC-0 residues mutated in this study are depicted by the colored and space-filled corresponding CLC-1 residues (in parenthesis): Blue, C212 (C277 of CLC-1); Grey, V490 (I556); Green, Y512 (Y578). The “E-gate” residue, E166 of CLC-0 (E232 of CLC-1), is also shown as a space-filled residue (red). (A) Stereo-view of hCLC-1 structure viewed from within membrane phospholipids (side view). Curved arrows depict the ion permeation pathways. Dotted lines indicate the extracellular and intracellular edges of lipid membranes. (B) Stereo-view of hCLC-1 viewed from the cytosolic side.

Fig 2. Voltage dependence of current activation of WT and mutant CLC-0.

(A) Voltage protocol (protocol I) for recordings. A full protocol consists of 12 recording sweeps. One sweep includes a prepulse voltage step at +60 mV (50 ms) followed by one of the various test voltage steps (70 ms) from +60 mV to -160 mV in -20 mV voltage steps, and followed by a tail voltage step of 50 ms at 0 mV (colored in black) or -100 mV (colored in red). Vertical dashed lines are used to mark the switch of the voltage steps (for example, between prepulse voltage step and the test voltage step, etc.). The voltage of the inter-sweep interval (ISI) was 0 mV and the duration was 1 or 4 sec. (B) Activation of WT CLC-0 and V490W mutant at pH_i = 7.4, using the voltage protocol shown in A. ISI was 4 sec. Dash line: zero-current level. Notice the inverted voltage-dependent activation in the mutant. (C) Activation of WT CLC-0 at pH_i = 5.5. ISI’s are 4 and 1 sec for the recording on the left and right, respectively. Notice the inverted voltage activation of WT CLC-0 in low pH_i.

Fig 3. Inhibition of WT CLC-0 by intracellular H⁺.

(A) Voltage protocol (protocol II) used for the experiments. (B & C) Inhibition of the CLC-0 current by an intracellular acidic solution (pH_i = 5). Circles represent the current measured at the steady state of the +60 mV voltage step as indicated by the downward arrows shown in A. ISI = 4 s and 1 s for the experiments in B and C, respectively. Notice the incomplete inhibition when ISI = 1 sec. Insets show traces for recording sweeps at the indicated time points.

Fig 4. Kinetics of the current inhibition and recovery of WT CLC-0 upon switching pH_i.

Voltage protocol was as that shown in Fig 3A. All currents measured at +60 mV were normalized to that obtained right before the application of low pH_i solutions. (A) Inhibition of WT CLC-0 currents by various low pH_i solutions. ISI = 4 s. The numbers above the horizontal lines (teal and red colors) indicate the values of pH_i. (B) Time constants of the inhibition (red squares) plotted against the values of pH_i (and thus [H⁺]_i). Time constants of current recovery (at pH_i = 7.4) are also plotted (sea green circles). Results were obtained from recordings like those shown in A (n = 3–6).

Fig 5. Kinetic analyses of current inhibition and recovery of the H⁺-induced WT CLC-0 inhibition.

Experiments were performed with protocol III on excised inside-out patches. Voltages were held constant throughout the recording sweep during which the intracellular solutions with different pH were switched. (A) Current inhibition and recovery at ±40 mV. The numbers above the dashed horizontal lines (red and sea green colors) indicate the values of pH_i. Fitted exponential decay curves (red) are superimposed with the recording traces in black. (B) Time constants of inhibition at three voltages (τ_inh) plotted against [H⁺]_i (or pH_i). The time constant of the slow-gate closure at pH_i = 7.4 at room temperature (measured separately) is shown by open square in sea green color (n = 5–8). (C) Voltage dependence of the inhibition time constant (τ_inh) (n = 5–9). (D) Current recovery time constants (τ_rec) against membrane voltages (n = 6).

Fig 6. Comparison of low pH_i-induced inhibitions between WT CLC-0 and the C212S mutant.

(A) Current inhibition of WT CLC-0 and C212S mutant at +20 mV by pH_i = 5.5. (B) Current inhibition of WT CLC-0 and C212S mutant at +20 mV by pH_i = 4.5. (C) Remaining current fraction (I/I_max) of WT CLC-0 and C212S mutant against [H⁺]_i (or pH_i) (n = 4–7). The steady-state current (I) was measured, respectively, at 5 s and 3 s (indicated by wine-colored arrows in A & B, respectively) after applying the pH_i 5.5 and pH_i 4.5 solutions. I_max was the current measured immediately before the low-pH_i solution was applied (teal color arrows in A & B).

Fig 7. Comparing the temperature dependence of low pH_i-induced inhibitions between WT CLC-0 and the C212S mutant.

(A) Inhibitions of WT CLC-0 by a solution with pH_i = 5.5 at three temperatures. (B) Inhibitions of the C212S mutant by a solution with pH_i = 4.5. All recording traces in A & B were obtained at V_m = ±20 mV. (C) Time constants of H⁺ inhibition of WT CLC-0 and the C212S mutant plotted against temperature (n = 5–11).

Fig 8. Comparing the voltage dependence of low pH_i-induced inhibition between WT CLC-0 and the C212S mutant.

(A) Recording traces showing the current inhibition induced by a solution with pH_i = 4.5 in WT CLC-0 and C212S. V_m = -40 mV. Values of the pH_i were shown below the colored horizontal lines. The values of τ_inh and τ_rec were obtained by fitting the current inhibition process (region shaded in pink color) and the current recovery process (region shaded in light blue) to single-exponential functions. (B) Voltage dependence of τ_inh of WT CLC-0 and C212S. All data points were obtained from the inhibition induced by a solution with pH_i = 4.5 (n = 4–9). (C) Voltage dependence of τ_rec of WT CLC-0 and C212S after pH_i = 4.5 was switched back to pH_i = 7.4 (n = 4–6). At positive voltages, current recovery was observed in the C212S mutant but not in WT CLC-0.

Fig 9. Correcting the inverted voltage-dependent opening in the V490W mutant by mutations that inhibit slow-gate closure.

(A) Comparing the inhibitions of WT CLC-0 and the Y512A mutant by solutions with pH_i = 5.5 (upper panel) and 4.5 (lower panel). (B) Remaining current fractions of WT CLC-0 and the Y512A mutant after the current inhibition by various [H⁺]_i (n = 5–6). (C) Recording traces of the double mutants V490W/C212S (left) and V490W/Y512A (right) obtained using the experimental protocol I with the tail step voltage at -100 mV. In both recordings, pH_i = 7.4. (D) Relative P_o of WT CLC-0, three mutants, C212S, V490W/C212S and V490W/Y512A. All data were obtained at pH_i = 7.4 (n = 3).