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Figure 1. Location of the residue D76 mapped on the structure of ClC-ec1 (PDB entry: 1OTS)A, dimeric structure of ClC-ec1 viewed from the membrane plane (extracellular side above and cytoplasmic side below). The two subunits are shown in green and cyan. Residue E148 (corresponding to E211 in ClC-5) is coloured in blue, D54 (corresponding to D76 of ClC-5) is shown in red and E203 (corresponding to E268 of ClC-5) in orange. Cl− anions bound to Scen and Sint are shown in magenta. B, expanded representation of the anion permeation pathway for one of the subunits. The position of the three binding sites, Sext, Scen and Sint, is also indicated by horizontal dashed lines. C, same representation as in B with a top view perpendicular to the membrane. In B and C, some transmembrane helices were removed for clarity.
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Figure 2. pH dependence of D76H and WTRepresentative current recordings for D76H (upper traces) and WT ClC-5 (lower traces) at pH 7.3 and 5.3. Voltages of the test pulse ranged from 120 to –80 mV. Here and in all figures with current traces, the dashed line represents the zero current level.
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Figure 3. pH dependence of inward tail currentsRepresentative current recordings for D76H (upper traces) and WT ClC-5 (lower traces) at some of the pHext values tested (7.3, 6.3, 5.3, 4.3), obtained with a voltage protocol comprising a pre-pulse and a post-pulse to 100 mV.
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Figure 4. Chloride dependence of inward tail currentsRepresentative current recordings for D76H (upper traces) and WT ClC-5 (lower traces) at different [Cl−]ext (10, 30, 100 and 300 mm). pHext is 5.3.
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Figure 5. Dependence of the inward tail currents of D76H on pHext and [Cl−]extA, changes in the reversal potential as a function of pHext are presented as ΔVrev, obtained as the difference between the reversal potential value at each pH and the value obtained at pH 5.3. Data are presented for pH 4.3 (n= 7), 4.8 (n= 7), 5.3 (n= 12), 6.3 (n= 5), 6.8 (n= 5), 7.3 (n= 5) and 8 (n= 6). Measurements were performed at 100 mm[Cl−]ext. The full, dashed-dotted, dashed and dotted lines represent the theoretical expectation for the changes in the reversal potential for transporters with 2 : 1, 3 : 1 or 1 : 1 Cl−/H+ stoichiometry or a pure proton conductance, respectively, under the same pH conditions. ΔVrev values calculated do not depend on assumptions on [Cl−]int (eqn (3)). B, the reversal potential as a function of [Cl−]ext is presented as the difference between the reversal potential value at each Cl− concentration and the value obtained at 100 mm[Cl−]. Data are presented for [Cl−] of 10 mm (n= 8), 30 mm (n= 7), 100 mm (n= 7) and 300 mm (n= 6). Full, dashed-dotted, dashed and dotted lines have the same meaning as in A. ΔVrev values calculated do not depend on assumptions on pHint (eqn (3)). Error bars are mostly smaller than symbols.
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Figure 6. Dependence of the inward tail currents on pHintA, representative inward tail currents at the indicated pHint values (5.3, 7.3, 9.3) from three different oocytes. [Cl−]ext is 110 mm and pHext is 5.8. Currents shown are the average of five traces. B, mean values of the reversal potential at pHint 7.3 (n= 5) and 9.3 (n= 3). The full, dashed-dotted, dashed and dotted lines represent the theoretical expectation for the changes in the reversal potential for transporters with a 2 : 1, 3 : 1 or 1 : 1 Cl−/H+ stoichiometry or a pure proton conductance, respectively, derived from eqn (3).
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Figure 7. Dependence of the inward tail currents on [Cl−]intA, representative inward tail currents at the indicated [Cl−]int (104 and 21 mm). [Cl−]ext is 110 mm and pHext is 5.8. Currents shown are the average of five traces. B, mean values of the reversal potential at [Cl−]int = 104 (n= 5) and 21 mm (n= 4). The full, dashed-dotted, dashed and dotted lines represent the theoretical expectation for the changes in the reversal potential for transporters with a 2 : 1, 3 : 1 or 1 : 1 Cl−/H+ stoichiometry or a pure proton conductance, respectively, derived from eqn (3). Error bars are smaller than symbols.
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Figure 8. Voltage dependence of gating of D76H as a function of pHextA, representative current traces at the indicated pHext from one oocyte. Red lines represent the mono-exponential fit of the tail currents elicited by a post-pulse to 100 mV. The amplitude of the tail currents at the onset of the post-pulse was extrapolated from the fit. These values were normalized with the parameter Imax derived from the fit with eqn (1) to obtain the current–voltage (I–V) relationships. B, I–V relationships for the oocyte shown in A. Full lines are the fit of the data with a Boltzmann function (eqn (1)) providing estimates for V1/2 and z of 21, 39, 89, 141 mV and 1.1, 1.0, 0.8 and 0.5, respectively, for pH 7.3, 6.3, 5.3 and 4.3. C. mean values of the difference between V1/2 calculated at pH 4.3 (n= 6), 6.3 (n= 5), 7.3 (n= 7) and that calculated at pH 5.3 (n= 10) as a function of pHext. The full line represents the fit of the data with a linear function with a slope of 30.7 ± 6.4 per pH unit.
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Figure 9. Comparison of the transient currents of E268A and D76H–E268A at different [Cl−]extA, representative current recordings from two oocytes, one expressing E268A (upper panel) and one expressing D76H–E268A (lower panel) at the indicated [Cl−]ext. B and C, average values for V1/2 and z, respectively, obtained from the Boltzmann fit of the Q–V relationship for [Cl−]ext of 0 mm (a contaminating concentration of 80 μm was assumed, see Zifarelli et al. 2012) (n= 6), 3 mm (n= 7), 10 mm (n= 6), 30 mm (n= 7) and 100 mm (n= 6) (eqn (3) and Supplemental material).
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Figure 10. Schematic model of the transport cycle of ClC-5 including a gating mechanismThe hypothetical model is based on the transport cycle suggested for ClC-Cm by Feng et al. (2010) with the addition of state (c). The model is not intended to provide a realistic description of the ClC-5 transport cycle, but rather to guide a mechanistic interpretation of the results. Circles represent Cl− ions; the shaded box represents the branch of the transport cycle that is accessible to the E268A mutant. From any of the states composing the transport cycle, indicated collectively by a brace, ClC-5 can transition to the inactive state I in which it is not able to transport. Forward and backward transitions to state I are voltage and pH dependent. In state (a), the side chain of E211 is unprotonated and the transporter has all the binding sites occupied by Cl− ions. In state (b), the side chain of E211 moves to occupy Sext and one Cl− ion moves intracellularly. In state (c), the side chain of E211 moves to occupy Scen and this is associated with the transport of another Cl− ion, whereas Sext is transiently empty but binds an extracellular Cl− ion in state (d). From this conformation E211 can be protonated by an intracellular proton (state e) and can move outwardly (state f), giving way to Cl− binding from the extracellular space (state g).
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