
Figure 1. Internal pH dependence of ClC0 fast gating. The figure illustrates the pH dependence of raw currents (A–E) and the analysis performed to extract the gating parameters (F and G). All data are from one patch expressing the C212S mutant. The symbols in F represent the (absolute values of the) initial tail currents at the tail pulse to −140 mV plotted as a function of the test pulse voltage. The symbols in G are the steadystate currents measured at the end of the test pulse. Lines in F are fits of Eq. 1 with the following parameters: pH 6.3, V1/2 = −142 mV, z = 1.9, pmin = 0.81; pH 7.3, V1/2 = −93 mV, z = 0.87, pmin = 0.19; pH 8.3, V1/2 = −30 mV, z = 0.99, pmin = 0.04; pH 9, V1/2 = 10 mV, z = 0.87, pmin = 0. Lines in G are fits of Eq. 2 with the following parameters: pH 7.3, G = 6.1 nS, V1/2 = −91 mV, z = 0.85, pmin = 0.15; pH 8.3, G = 6.1 nS, V1/2 = −31 mV, z = 1.08, pmin = 0.04; pH 9, G = 5.9 nS, V1/2 = 7 mV, z = 1.04, pmin = 0.004; pH 10, G = 4.6 nS, V1/2 = 88 mV, z = 0.77, pmin = 0.008.


Figure 2. Dependence of the voltage of halfmaximal activation on internal pH. Mean values of V1/2 are plotted as a function of pH. Error bars indicate SD. The straight line has a slope of 46 ± 2 mV per pH unit, corresponding to 20 mV per efold change of the H+ concentration.


Figure 3. Indication of the mutated amino acids on a topological model of ClC0 based on the crystal structure of ClCec1 (Dutzler et al., 2002).


Figure 4. pH dependence of mutant E166A. Shown are current traces from an insideout patch at control pH and at pH 11. Note the timedependent current decrease at negative voltages. Similar results were obtained in two other patches.


Figure 5. Kinetic analysis of fast gating. In A are shown typical traces (from a patch at pH 9) used for the determination of the single exponential relaxation time constants. In the right panel the capacitive transient has been blanked for clarity. Red lines are single exponential fits with τ = 0.79 ms (−140 mV) and τ = 0.18 ms (+140 mV). In B are plotted mean values for the time constants at various pH as a function of voltage.


Figure 6. Opening and closing rate constants at different pH. From the time constants (Fig. 5) and the steadystate open probability (see Fig. 1) the opening rate constants (A) and the closing rate constants (B) were calculated according to Eq. 3. For clarity, error bars are not shown.


Figure 7. Kinetic analysis of fast gating at low (14 mM) Cl−int. (A) Typical traces (from a patch at pH 9) used for the determination of the single exponential relaxation time constants. In the right panel the capacitive transient has been blanked for clarity. Red lines are single exponential fits with τ = 0.37 ms (−140 mV) and τ = 0.22 ms (+140 mV). (B) Plots of mean values for the closing rate constant determined as in Fig. 6 at various pH as a function of voltage.


Figure 8. pH dependence of the closing rate constants—first models. (A) Plots of the values for the closing rate constant (same values as in Fig. 6) as a function of pH with different symbols for different voltages (for clarity, values are shown only for a few representative voltages: black circles, −180 mV; pink triangles down, −120 mV; green diamonds, −80 mV; red triangles up, −40 mV; blue squares, 20 mV; gray circles, 60 mV). Solid lines are fits of Eq. 5 with the resulting pKO and β0 reported in B and C, respectively, as filled circles. The solid line in C is a fit of the filled circles to an exponential function of the form β0 = β0(0)*exp(z*VF/(RT)), where F is Faradays constant, R the gas constant, T the absolute temperature, β0(0) the value of β0 at 0 mV, and z the “slope” of the curve, resulting in z = 0.28. Dashed lines in A are fits of Eq. 7 with the resulting pKO and μ reported in B and C, respectively, as open circles.


Figure 9. Model of pHdependent gating of ClC0 involving water dissociation. In A, the model is schematically drawn (see text for details). (B and C) Closing rate constants at 104 mM Cl− (B) and 14 mM Cl− (C) (data from Fig. 6 B and Fig. 7 B, respectively; circles, pH 7.3; squares, pH 8.3; triangles up, pH 9; triangles down, pH 10) together with a best fit of Eq. 8 with the following parameters: β(0) = 6003 s−1, zβ = 0.31, KCl(0) = 16.6 mM, KOH(0) = 46 μM, zCl = 0. (D) A hypothetical model of how extracellular Cl− ions could facilitate gate opening. The initially protonated state (indicated by *) is metastable and requires the occupation of the external binding site by a Cl− ion for channel opening.


Figure 10. Fast gating relaxations in D2O. Representative current traces from an inside out patch measured in the standard H2O solution (A) or in D2O (B) at pH/pD 8.3. The extracellular solution was in H2O. The pulse protocol consisted of a prepulse to −140 mV, test pulses ranging from 140 to −160 mV, and a tail pulse to 100 mV. The right panels show a zoom of the pulses to 100 mV, superimposed with a monoexponential fit (red line).


Figure 11. Opening and closing rate constants in D2O. Opening (A) and closing (B) rate constants were determined as in Fig. 6. Since slightly different solutions were employed in these experiments (see Materials and methods), the rate constants in H2O represent the values obtained from the same patches in which we tested the effect of D2O. (C) The ratio of the opening rate constants at pH/pD 8.3 in H2O and D2O. (D) The ratio of the closing rate constants at pH/pD 8.3 in H2O and D2O.
