Chen MF , Chen TY .

	Scheme S1.
	Figure 1. Quasi steady-state inactivation curves of the WT ClC-0 and the C212S mutant at different pH values. (A) Whole oocyte currents of the WT and C212S at three external pH. See materials and methods for the voltage protocol to examine the slow-gate inactivation. Dotted lines represent zero-current level. (B) Quasi steady-state inactivation curve of the WT (left, n = 3) and the C212S mutant (right, n = 4) at pH 7.6 (circles), 9.6 (triangles), and 5.6 (squares).
	Figure 2. Effect of the external Cl− concentration on the fast gating of the WT channel and the C212S mutant. (A) Whole-cell current of the WT- and C212S-injected oocytes at 98 and 4 mM [Cl−]o. Voltage protocol is as described in materials and methods with a maximal depolarized voltage of +120 mV. (Insets) Expanded current traces corresponding to those within the squares to demonstrate the initial current of the fast gating relaxation at −100 mV. (B) Steady-state Po-V curve of WT and C212S at various [Cl−]o. Symbols are as follows: ○, 98 mM; Δ, 30 mM; ▿, 15 mM; and □, 4 mM. Solid curves were drawn according to a Boltzmann equation: Po = Pmin + (1 − Pmin)/[1 + exp(−zF(V − V1/2)/RT)], with z = 0.8–1.2, Pmin = 0.03–0.05. V1/2's were as follows: for WT, −91 (98 mM), −60 (30 mM), −42 (15 mM), and −14 mV (4 mM); for C212S, −79 (98 mM), −56 (30 mM), −39 (15 mM), and −12 mV (4 mM).
	Figure 3. External pH effect on the macroscopic current of the WT and the C212S mutant. The recording traces at different pH were from the same oocyte. Voltage protocols are the same as in Fig. 2 A except that a maximal depolarizing test voltage of +80 mV was used. Dotted lines are the zero current level.
	Figure 4. Fast-gating properties of the WT and C212S at different external pH values. All parameters were derived from macroscopic current recordings such as those shown in Fig. 3. (A) Effect of pH on the steady-state Po-V curve. All data were normalized to the initial tail current after a test pulse of +80 mV. (B) Effect of pH on the opening rate of the channels. (C) Effect of pH on the closing rate of the channel. Opening and closing rates were calculated according to and . Symbols and the pH were as follows: □, 5.1; ▪, 5.6; Δ, 6.1; ▴, 6.6; ○, 7.1; •, 7.6; and ▿, 9.6. Data points were connected by short straight lines.
	Figure 5. Single-channel recordings of C212S at different external pH. (A) Single-channel recording traces at the indicated external pH. Dotted lines are the assigned three current levels. C, closed state of the channel; O, fully open state of the channel. (B) All points amplitude histograms compiled from 30 s (pH 9.6), 30 s (7.6), 40 s (6.6), and 40 s (5.6) recording traces containing the example segments shown in A. The measured state probabilities (f0, f1, and f2) are as follows: (pH 9.6) 0.298, 0.491, and 0.211; (pH 7.6) 0.232, 0.496, and 0.272; (pH 6.6) 0.071, 0.402, and 0.527; (pH 5.6) 0.010, 0.214, and 0.776, resulting in the calculated Po values shown in A. Assuming a binomial distribution, the predicted state probabilities (f0, f1, and f2) calculated from Po are as follows: for pH 9.6, 0.295, 0.496, and 0.209; for pH 7.6, 0.230, 0.499, and 0.270; for pH 6.6, 0.074, 0.396, and 0.530; and for pH 5.6, 0.014, 0.206, and 0.780. (C) Dwell-time distributions of the events at the three current levels. Same traces as in B. Δ, ▪, and ○ represent the closed (level 0), intermediate (level 1), and fully open (level 2) current levels, respectively. The fitted time constants (τ0, τ1, and τ2) are as follows (in ms): for pH 9.6, 19.5, 18.8, and 17.6; for pH 7.6, 17.5, 19.9, and 18.7; for pH 6.6, 6.6, 10.9, and 19.8; and for pH 5.6, 1.8, 4.3, and 20.3. As discussed in materials and methods, because there are only 140 closed events at pH 5.6 in this analysis and their durations are heavily affected by the cutoff frequency, the estimate of τ0 at this acidic condition may have a relatively large error.
	Figure 6. Fast gating properties of single C212S channels at different external pH. (A) Steady-state open probability. (B) Opening rate of the fast gate. (C) Closing rate of the fast gate. All rate constants were calculated according to and . External pHs were as follows: ▿, 5.6; Δ, 6.6; □, 7.6; and ○, 9.6.
	Figure 7. External H+ modulation of the fast gate Po-V curve of C212S at saturating and nonsaturating [Cl−]o. Data were derived from macroscopic current recordings at pH 7.6 (○) and 5.6 (□). (A) Comparison of the Po-V curves under different pH at 15 mM [Cl−]o (n = 3). (B) Comparison as performed in A at 300 mM [Cl−]o (n = 3–4). Data points were connected with short straight lines.
	Figure 8. External H+ activates the hyperpolarization-favored opening process. (A) Modeling the fast-gate opening rate according to Fig. 1. The solid curve was generated according to , using the values listed in Table II of Chen and Miller 1996. See discussion for the value of each parameter. Dotted curves were synthesized by increasing the value of α1(0) as shown on the left of each curve. (B and C) Examination of the fast-gate by the sum of two opening processes. Opening rate data were taken from macroscopic (B) and single-channel recordings (C) as those shown in Fig. 4 B and 6 B, respectively. The dataset at each external pH was fitted to constrained with a shared value of γ(0). Only α1(0) and γ(0) were allowed to vary in the simultaneous multiple-curve fitting process. All the other parameters were the same as those in A. The fitted γ(0) were 590 s−1 and 340 s−1 in B and C, respectively. (D) The fitted α1(0) from B (□) and C (X) as a function of external pH. Data points from macroscopic recordings were further fitted to a logistic function, A1+ (A2 − A1)/(1 + [H+]o/Ka), where Ka is the dissociation constant of the protonation site for H+. The minimal (A1) and maximal (A2) α1(0) were 0.85 s−1 and 31.6 s−1, respectively. The fitted Ka is 4.7 × 10−6 M, corresponding to pKa = 5.3.

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