Chen TY et al. (2003), Electrostatic control and chloride regulation o...

XB-ART-4497

J Gen Physiol 2003 Nov 01;1225:641-51. doi: 10.1085/jgp.200308846.

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Electrostatic control and chloride regulation of the fast gating of ClC-0 chloride channels.

Chen TY , Chen MF , Lin CW .

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The opening and closing of chloride (Cl-) channels in the ClC family are thought to tightly couple to ion permeation through the channel pore. In the prototype channel of the family, the ClC-0 channel from the Torpedo electric organ, the opening-closing of the pore in the millisecond time range known as "fast gating" is regulated by both external and internal Cl- ions. Although the external Cl- effect on the fast-gate opening has been extensively studied at a quantitative level, the internal Cl- regulation remains to be characterized. In this study, we examine the internal Cl- effects and the electrostatic controls of the fast-gating mechanism. While having little effect on the opening rate, raising [Cl-]i reduces the closing rate (or increases the open time) of the fast gate, with an apparent affinity of >1 M, a value very different from the one observed in the external Cl- regulation on the opening rate. Mutating charged residues in the pore also changes the fast-gating properties-the effects are more prominent on the closing rate than on the opening rate, a phenomenon similar to the effect of [Cl-]i on the fast gating. Thus, the alteration of fast-gate closing by charge mutations may come from a combination of two effects: a direct electrostatic interaction between the manipulated charge and the negatively charged glutamate gate and a repulsive force on the gate mediated by the permeant ion. Likewise, the regulations of internal Cl- on the fast gating may also be due to the competition of Cl- with the glutamate gate as well as the overall more negative potential brought to the pore by the binding of Cl-. In contrast, the opening rate of the fast gate is only minimally affected by manipulations of [Cl-]i and charges in the inner pore region. The very different nature of external and internal Cl- regulations on the fast gating thus may suggest that the opening and the closing of the fast gate are not microscopically reversible processes, but form a nonequilibrium cycle in the ClC-0 fast-gating mechanism.

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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 Clâ 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.
	Figure 2. . Gating effects of [Clâ]i on the WT channel. (A) Single-channel recordings and dwell-time analyses of the WT channel. (Left) Recording traces at various [Clâ]i as indicated. Vm = â110 mV, and [Clâ]o = 120 mM. (Right) Dwell-time histograms from continuous recordings containing the 6-s traces on the left. Symbols are: â¡, closed level; Î, middle level; and â¢, 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 [Clâ]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 [Clâ]i (in mM) are: â¡, 120; â¢, 300; Î, 600; âª, 1,200; â, 2,400.
	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 = â110 mV; [Clâ]o = 120 mM; [Clâ]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: â¡, WT; â¢, K519C; Î, K519E.
	Figure 4. . Dependence of the fast-gate closed (Ïc) and open durations (Ïo) on internal Clâ. Ïo and Ïc were calculated according to Eqs. 3a and b, respectively. Vm = â110 mV. (A) Ïc as a function of internal Clâ activity. Symbols are: squares, WT; circles, K519C; triangles, K519E. Data points are connected by straight lines. (B) Ïo as a function of internal Clâ activity. Solid curves were drawn according to Eq. 4. The fitted parameters (Ïmin, Ïmax, 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 Ïmax, and K1/2 were fixed as those for the WT channel. The fitted values of Ïmin in these two channels were 28.4 and 42.5 ms, respectively.
	Figure 5. . Effects of permeant and nonpermeant ions on the gating and permeation properties of WT ClC-0. (A) Blocking effects of Brâ on the conductance of the WT channel. Squares and the solid curve are the conductance-[Clâ] curve for the WT channel in the absence of Brâ. Circles are obtained in 300 mM [Clâ]i plus 3, 10, 30, 60, 300, and 900 mM Brâ, 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 â gmin)/(1+([Brâ]/K1/2), where gmax (=12.17 pS) is the conductance at [Clâ]i = 300 mM in the absence of Brâ. The fitted gmin and K1/2 were 5.6 pS and 23.8 mM, respectively. (B) Gating effects of Brâ and SO42â on the WT channel. Ïo 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 [Clâ]i. Open circles were from the Brâ experiments as those described in A. Open triangles were from recordings at 120 mM [Clâ]i plus 180 and 480 mM SO42â. All experiments shown in A and B were from recordings at the membrane potential of â110 mV.
	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 = â110 mV. [Clâ]i = 600 mM. (B) Averaged closed durations of the three channels shown in A as a function of internal Clâ 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 Clâ 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.
	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 = â90 mV and [Clâ]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 [Clâ]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 = â90 mV and [Clâ]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.
	Figure 8. . Internal Clâ titration on the closed (Ïc) and the open durations (Ïo) of the mutants at the selectivity filter. (A) Single-channel recording traces for the mutants Y512F and S123T at 300 and 2400 mM [Clâ]i. Vm = â110 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 [Clâ]i on Ïc of Y512F. Data points were calculated from opening rate at â110 mV according to Eq. 3b. The values of Ïc 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 [Clâ]i on Ïo. Data at â110 mV were used to calculate Ïo 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 Ïmin, Ïmax, and K1/2, respectively.

References [+] :

Bauer, Completely functional double-barreled chloride channel expressed from a single Torpedo cDNA. 1991, Pubmed, Xenbase