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Figure 1. . Stereo view of the inner pore region of the bacterial ClC channel. Extracellular side of the molecule is on top. The labeling outside and inside the parentheses represents residues of the bacterial and the Torpedo channel, respectively. The two spheres represent the two Cl− ions at Scen (top) and Sint (bottom). To reveal the relations of these residues with respect to the glutamate gate, E148 (E166 of ClC-0) is shown in black on top. The dotted, curved arrow roughly represents the ion permeation pathway. Ribbons represent helices D and R where these residues are located. Structural coordinates were taken from Protein Data Bank with the accessing code 1OTS.
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Figure 2. . Gating effects of [Cl−]i on the WT channel. (A) Single-channel recordings and dwell-time analyses of the WT channel. (Left) Recording traces at various [Cl−]i as indicated. Vm = −110 mV, and [Cl−]o = 120 mM. (Right) Dwell-time histograms from continuous recordings containing the 6-s traces on the left. Symbols are: □, closed level; Δ, middle level; and •, fully open level. The length of the analyzed trace and the number of events in each analysis at closed, middle, and fully open levels, respectively, are: (120 mM) 62 s and 1,147, 1,572, 426; (600 mM) 60 s and 659, 1,251, 593; (2,400 mM) 52 s and 197, 644, 449. (B) Averaged opening rate (left) and closing rate (right) of WT ClC-0 under different [Cl−]i. The rate parameters were calculated according to Eqs. 2a and b, and plotted (in logarithmic scale) as a function of the membrane potential Vm. Symbols for [Cl−]i (in mM) are: □, 120; •, 300; Δ, 600; ▪, 1,200; ○, 2,400.
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Figure 3. . Mutational effects of residue K519 on the fast gating properties of ClC-0. (A) Single-channel recordings and dwell-time analyses for the WT channel and K519C and K519E mutants. Vm = −110 mV; [Cl−]o = 120 mM; [Cl−]i = 600 mM. Symbols in the dwell time histograms represent the same current levels as indicated in Fig. 2 A. The length of the analyzed trace and the numbers of events (closed, middle and fully open levels) for each analysis were: (WT), 50 s and 480, 1,010, 532; (K519C) 92 s and 457, 1,452, 997; (K519E) 112 s and 446, 1,561, 1,118. (B) Averaged opening and closing rates for the three channels shown in A as a function of voltage. Experimental conditions are as in A. Symbols are: □, WT; •, K519C; Δ, K519E.
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Figure 4. . Dependence of the fast-gate closed (τc) and open durations (τo) on internal Cl−. τo and τc were calculated according to Eqs. 3a and b, respectively. Vm = −110 mV. (A) τc as a function of internal Cl− activity. Symbols are: squares, WT; circles, K519C; triangles, K519E. Data points are connected by straight lines. (B) τo as a function of internal Cl− activity. Solid curves were drawn according to Eq. 4. The fitted parameters (τmin, τmax, and K1/2) for the WT channel (squares) were: 3.8 ms, 95.5 ms, and 1,313 mM. In fitting the data points for K519C and K519E channels, the values of τmax, and K1/2 were fixed as those for the WT channel. The fitted values of τmin in these two channels were 28.4 and 42.5 ms, respectively.
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Figure 5. . Effects of permeant and nonpermeant ions on the gating and permeation properties of WT ClC-0. (A) Blocking effects of Br− on the conductance of the WT channel. Squares and the solid curve are the conductance-[Cl−] curve for the WT channel in the absence of Br−. Circles are obtained in 300 mM [Cl−]i plus 3, 10, 30, 60, 300, and 900 mM Br−, respectively. Conductance in various ionic conditions was determined from the slope of the single-channel i-V curve (Chen and Chen, 2003). Dotted curve represents the best fit to an equation: g = gmin + (gmax − gmin)/(1+([Br−]/K1/2), where gmax (=12.17 pS) is the conductance at [Cl−]i = 300 mM in the absence of Br−. The fitted gmin and K1/2 were 5.6 pS and 23.8 mM, respectively. (B) Gating effects of Br− and SO42− on the WT channel. τo was estimated from the closing rate according to Eq. 3a. Solid squares and the solid curve were the same as those from the WT channel in Fig. 4 except the data were plotted against [Cl−]i. Open circles were from the Br− experiments as those described in A. Open triangles were from recordings at 120 mM [Cl−]i plus 180 and 480 mM SO42−. All experiments shown in A and B were from recordings at the membrane potential of −110 mV.
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Figure 6. . Functional role of E127 in the fast gating of ClC-0. (A) Single-channel recording traces and dwell-time histograms of E127Q/K519, E127Q/K519C, and E127Q/K519E. Symbols in the histograms represent the same current levels as those described in Fig. 2 A. Vm = −110 mV. [Cl−]i = 600 mM. (B) Averaged closed durations of the three channels shown in A as a function of internal Cl− activity. Symbols are: squares, E127Q/K519; circles, E127Q/K519C; triangles, E127Q/K519E. Data points are connected by straight lines. Dotted lines are the same as those straight lines in Fig. 4 A. (C) Averaged open durations as a function of internal Cl− activity. Symbols are the same as in B. Dotted curves are the same as the solid curves in Fig. 4 B. Note that in the presence of E127Q mutation, the mutation effects from varying the charge at position 519 were nearly eliminated.
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Figure 7. . Increase of the fast-gating transition rate by increasing positive potential in the pore. (A) Single-channel recordings of WT, I515K and D513S mutants at Vm = −90 mV and [Cl−]i = 600 mM. Dotted lines represent zero-current level. (B) Comparison of the averaged opening (open bars) and the closing rates (filled bars) among the WT channel, I515K and D513S. Vm and [Cl−]i are as described in A. Both the opening and closing rates of the mutants are significantly larger than those of the WT channel. (C) Placing positive charge at position 127 also speeds up the fast gating. Recording traces of the double mutants were taken at Vm = −90 mV and [Cl−]i = 300 mM. Note that the only structural difference of these two double mutants was the mutation at position 127. (D) Comparison of the opening (open bars) and closing rates (filled bars) of the WT channel and the double mutants involving positions 127 and 519. The opening rates among three channels are not significantly different. The closing rate of E127K/K519E is significantly larger than that of the WT channel and of the E127Q/K519E double mutant.
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Figure 8. . Internal Cl− titration on the closed (τc) and the open durations (τo) of the mutants at the selectivity filter. (A) Single-channel recording traces for the mutants Y512F and S123T at 300 and 2400 mM [Cl−]i. Vm = −110 mV. For comparison, the traces of the WT channel at the same recording conditions are also shown. The vertical scale for the S123T traces (left and right) is expanded to better show the close-open transition of this channel. (B) Effects of raising [Cl−]i on τc of Y512F. Data points were calculated from opening rate at –110 mV according to Eq. 3b. The values of τc for WT, K519C and K519E connected by dotted lines are the same as those in Fig. 4 A. Those values for Y512F are shown as open circles. (C) Effects of raising [Cl−]i on τo. Data at −110 mV were used to calculate τo according to Eq. 3a. Closed circles represent the values for Y512F. Dotted curves were taken from Fig. 4 B. The solid curve was drawn according to a curve fitting to Eq. 4, with the fitted parameters of 12.9 ms, 182.1 ms, and 1334.9 mM for τmin, τmax, and K1/2, respectively.
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