Figure 5.

Gating properties of the disease associated mutations G212A and E267A ClC-5. (A) Averaged current–voltage relations constructed by measuring the steady-state current amplitudes at the end of voltage steps in the indicated range as depicted in Figures 3D–F for cells expressing either WT (n = 5), G212A (n = 6) or E267A (n = 11) ClC-5. Significant differences (for both mutants vs. WT ClC-5) at the level of 0.05 are indicated as stars. (B) Ratio between the off-gating charge (Q_off) and ionic currents (I) at +165 mV [n = 5.11, same cells as in (A)]. Significant differences at the level of 0.05 are indicated as stars. (C) Voltage dependence of the non-linear capacitances of WT and G212A and E267A ClC-5 determined by using lock-in based impedance measurements. The curves for E267A and WT ClC-5 exhibit the typical bell-shaped form and were fitted with the first derivative of a standard Boltzmann function with the peak corresponding to the half-maximal activation (E267A ClC-5 - V_0.5 = 153 ± 2 mV, n = 5; WT ClC-5 - V_0.5 = 128 ± 2 mV, n = 5). The curve for mutation G212A did not exhibit a peak within the investigated range and was not fitted. (D) Voltage dependence of the activation of mutation G212A WT as obtained from gating current measurements. The inset depicts a representative recording from WT ClC-5 measured with short pulses. The encircled region is additionally depicted enlarged to show the off-gating currents that were integrated to obtain the gating charge Q_off. The red lines represent Boltzmann fits to the data to obtain the half maximal voltage of activation (G212A and WT ClC-5, respectively, V_0.5 = 240 ± 10 mV, n = 4 and V_0.5 = 132 ± 6 mV, n = 4).