Zifarelli G et al. (2008), Intracellular proton regulation of ClC-0.

XB-ART-38450

J Gen Physiol 2008 Jul 01;1321:185-98. doi: 10.1085/jgp.200809999.

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Intracellular proton regulation of ClC-0.

Zifarelli G , Murgia AR , Soliani P , Pusch M .

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Some CLC proteins function as passive Cl(-) ion channels whereas others are secondary active chloride/proton antiporters. Voltage-dependent gating of the model Torpedo channel ClC-0 is modulated by intracellular and extracellular pH, possibly reflecting a mechanistic relationship with the chloride/proton coupling of CLC antiporters. We used inside-out patch clamp measurements and mutagenesis to explore the dependence of the fast gating mechanism of ClC-0 on intracellular pH and to identify the putative intracellular proton acceptor(s). Among the tested residues (S123, K129, R133, K149, E166, F214L, S224, E226, V227, C229, R305, R312, C415, H472, F418, V419, P420, and Y512) only mutants of E166, F214, and F418 qualitatively changed the pH(int) dependence. No tested amino acid emerged as a valid candidate for being a pH sensor. A detailed kinetic analysis of the dependence of fast gate relaxations on pH(int) and [Cl(-)](int) provided quantitative constraints on possible mechanistic models of gating. In one particular model, a proton is generated by the dissociation of a water molecule in an intrapore chloride ion binding site. The proton is delivered to the side chain of E166 leading to the opening of the channel, while the hydroxyl ion is stabilized in the internal/central anion binding site. Deuterium isotope effects confirm that proton transfer is rate limiting for fast gate opening and that channel closure depends mostly on the concentration of OH(-) ions. The gating model is in natural agreement with the finding that only the closing rate constant, but not the opening rate constant, depends on pH(int) and [Cl(-)](int).

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Species referenced: Xenopus
Genes referenced: gnas tbx2

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	Figure 1. Internal pH dependence of ClC-0 fast gating. The figure illustrates the pH dependence of raw currents (AâE) 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 â140 mV plotted as a function of the test pulse voltage. The symbols in G are the steady-state 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 = â142 mV, z = 1.9, pmin = 0.81; pH 7.3, V1/2 = â93 mV, z = 0.87, pmin = 0.19; pH 8.3, V1/2 = â30 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 = â91 mV, z = 0.85, pmin = 0.15; pH 8.3, G = 6.1 nS, V1/2 = â31 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 half-maximal 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 Â± 2 mV per pH unit, corresponding to 20 mV per e-fold change of the H+ concentration.
	Figure 3. Indication of the mutated amino acids on a topological model of ClC-0 based on the crystal structure of ClC-ec1 (Dutzler et al., 2002).
	Figure 4. pH dependence of mutant E166A. Shown are current traces from an inside-out patch at control pH and at pH 11. Note the time-dependent 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 Ï = 0.79 ms (â140 mV) and Ï = 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 steady-state 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) Clâint. (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 Ï = 0.37 ms (â140 mV) and Ï = 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 constantsâfirst 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, â180 mV; pink triangles down, â120 mV; green diamonds, â80 mV; red triangles up, â40 mV; blue squares, 20 mV; gray circles, 60 mV). Solid lines are fits of Eq. 5 with the resulting pKO and Î²0 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 Î²0 = Î²0(0)exp(zVF/(RT)), where F is Faradays constant, R the gas constant, T the absolute temperature, Î²0(0) the value of Î²0 at 0 mV, and z the âslopeâ of the curve, resulting in z = 0.28. Dashed lines in A are fits of Eq. 7 with the resulting pKO and Î¼ reported in B and C, respectively, as open circles.
	Figure 9. Model of pH-dependent gating of ClC-0 involving water dissociation. In A, the model is schematically drawn (see text for details). (B and C) Closing rate constants at 104 mM Clâ (B) and 14 mM Clâ (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: Î²(0) = 6003 sâ1, zÎ² = 0.31, KCl(0) = 16.6 mM, KOH(0) = 46 Î¼M, zCl = 0. (D) A hypothetical model of how extracellular Clâ ions could facilitate gate opening. The initially protonated state (indicated by *) is metastable and requires the occupation of the external binding site by a Clâ 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 â140 mV, test pulses ranging from 140 to â160 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.

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Accardi, Ionic currents mediated by a prokaryotic homologue of CLC Cl- channels. 2004, Pubmed