Drastic reduction of the slow gate of human muscle chloride channel (ClC-1) by mutation C277S

Abstract

1. Single channel measurements suggest that the human muscle chloride channel ClC-1 presumably has a double barrelled structure, with a fast single protopore gate and a slow common pore gate similar to that of ClC-0, the chloride channel from Torpedo. The single point mutation C212S has been shown to abolish the slow gating of ClC-0 locking the slow gate in the open state. In order to test the hypothesis that the slow gating process found in ClC-1 corresponds to the well characterised slow gate found in ClC-0 we investigated the gating effects in ClC-1 of the homologous mutation corresponding to C212S, C277S. 2. We found that the mutation C277S strongly reduced the slow component of macroscopic gating relaxations at negative and at positive voltages. 3. Time constants of the fast gating relaxations were not affected by the mutation but the minimal open probability of the fast gate at negative voltages was slightly reduced to 0.08 compared with the WT value of 0.22. 4. Additionally, we characterised the block of WT ClC-1 and mutant C277S by the S(-) enantiomer of CPB (2-(p-chlorophenoxy) butyric acid), and found that the block is practically unaffected by the mutation suggesting that CPB does not interact with the slow gate of ClC-1. 5. We conclude that the slow and fast gating processes of ClC-1, respectively, reflect the slow common pore gate and the single protopore gate of the double-barrelled ClC-1 channel.

Figure 1. Deactivation of mutant C277S fits a single exponential

A, I-V protocol. Following a 50 ms pulse to +60 mV, to fully activate the currents, the voltage was stepped to values increasing in 20 mV steps between -160 and +80 mV for 200 ms. Finally, the voltage was stepped to -140 mV for 20 ms to record the tail currents. B, WT ClC-1 currents elicited with the protocol shown in A. C, currents of mutant C277S. D, double logarithmic representation of the trace at -120 mV from B. Single (continuous line) and double (dashed line) exponential fits are also plotted. Parameters obtained with the single exponential fit are: τ_f = 19.7 ms, while the double exponential fit yielded τ_f = 12.3 ms, τ_s = 85.8 ms and C_f/C_s = 3.2. In the inset the same trace and fits are plotted in a linear representation. E, double logarithmic representation of the trace at -120 mV from C, single (continuous line) and double (dashed line) exponential fits are also plotted. Parameters obtained with the single exponential fit are: τ_f = 13.3 ms, while the double exponential fit yielded τ_f = 11.8 ms, τ_s = 32.9 ms and C_f/C_s = 8.7. In the inset the same trace and fits are plotted in a linear representation.

Figure 2. Voltage dependence of the time constants of mutant C277S

A, fast gating process time constant of mutant C277S obtained with envelope protocols. A 200 ms pulse to -140 mV is followed by a short pulse of varying duration to +100 mV (increasing in 10 μs steps). The patch is then hyperpolarised to -140 mV for 10 ms. The value of the initial current recorded upon repolarization back to -140 mV is evaluated with a single exponential fit of the trace at -140 mV. Only 1 trace out of 4 is plotted for clarity's sake. B, initial values of the current at -140 mV plotted as a function of the time spent at +100 mV, t_p. Dashed line (hardly visible because almost superimposed on the data points) is a double exponential fit, and the continuous line is a single exponential fit. C, voltage dependence of the time constant of the fast gate of WT ClC-1 (○) and of mutant C277S (•). Error bars represent s.e.m. (n≥ 3 for all voltages). WT values taken from Accardi & Pusch (2000). The time constants of the WT and mutant channel were not different (P > 0.20) at all voltages apart from -100, -40 and +20 mV where P > 0.05. At -60 and +60 mV the test indicated that the two were significantly different (P < 0.05), but the relatively small number of experiments at these voltages prevents a meaningful statistical evaluation.

Figure 3. Separation of fast and slow gating process open probabilities of mutant C277S

A, a 200 ms long pulse of increasing voltage from -140 to +100 mV in 20 mV steps is followed by a 20 ms repolarization to -140 mV. The initial part of the 200 ms prepulse is not shown. B, currents recorded when 200 ms pulses are followed by a short 200 μs pulse to +200 mV, prior to the 20 ms repolarization to -140 mV. The initial part of the 200 ms pulse is not shown. Dashed line represents zero current. C, open probabilities for the slow gate (SG) of WT ClC-1 (▵) and mutant C277S (▴). Continuous lines are the fits of the open probabilities with eqn (1). The values obtained from the mean of at least 7 different experiments are: V_½(SG) = -48 ± 4 mV; P₀ (SG) = 0.84 ± 0.01; z (SG) = 1.0 ± 0.1. Error bars represent s.e.m. (n = 7). WT values taken from Accardi & Pusch (2000). The residual open probability of the slow gating process in the WT and mutant channel are significantly different (P < 10⁻⁸). D, open probabilities for the fast gate (FG) of WT ClC-1 (○) and mutant C277S (•). Continuous lines are the fits of the open probabilities with eqn (1). The values obtained from the mean of at least 6 different experiments are: V_½(FG) = -73 ± 3 mV; P₀ (FG) = 0.08 ± 0.02; z (FG) = 1.1 ± 0.1. Error bars represent s.e.m. (n = 7). WT values are taken from Accardi & Pusch (2000).

Figure 4. Block of mutant C277S by S(-)-CPB

The stimulation protocol in A and B is similar to that described in Fig. 1A (but the tail voltage is -100 mV instead of -140 mV). A, control. B, current traces recorded in presence of 1 mm S(-)-CPB. Inset: slow deactivating component at -140 mV; the time constant of this component is τ_vs = 22.3 ms. C, initial tail currents at -100 mV are plotted as a function of the prepulse voltage in control conditions (•) and with 1 mm S(-)-CPB (▪). The continuous line is a fit with eqn (1) with the following parameters: V_½(control) = -68 mV; P₀ (control) = 0.04; z (control) = 1.2 and V_½(CPB 1 mm) = 23 mV; P₀ (CPB 1 mm) = 0.003; z (CPB 1 mm) = 0.94. D, comparison of the K_d for S(-)-CPB obtained from tail currents (○, •) and from steady-state current (□, ▪) for WT ClC-1 (○, □) and mutant C277S (•, ▪). The K_d was obtained from eqn (2). Error bars represent s.e.m. (n = 4 both for WT and C277S mutant). The K_d for WT and mutant are not different (P > 0.21 at all voltages except +20 and +40 mV where P > 0.05).