Mutations at the hydrophobic gate or site 1 alter the facilitation of the voltage dependence induced by extracellular SCN ⁻ MFs. The light blue lines in G–J plots are average WT values replotted from Fig. 5. (A, C, and E) Macroscopic currents recorded from cells expressing I641A and E702Q/E705Q channels. Each pair was obtained from the same cell dialyzed with 0 or 0.2 (I641A) or 12 (E702Q/E705Q) µM Ca²⁺ in the presence of extracellular Cl⁻ (black) and then in the presence of SCN⁻ (red). Scale bars are valid for both sets of traces. Gray lines indicate the zero-current levels. (B, D, and F) Voltage dependence of normalized conductance of I641A (B and D) and E702Q/E705Q (D) channels calculated from recordings obtained in the presence of the indicated SCN⁻ MF in B. For I641A in 0 Ca²⁺, I641A in 0.2 µM Ca²⁺, and E702Q/E705Q in 12 µM Ca²⁺ were n = 19, 23, and 24 (SCN⁻ MF = 0); 5, 9, and 7 (SCN⁻ MF = 0.25); 5, 8, and 10 (SCN⁻ MF = 0.5); 5, 5, and 6 (SCN⁻ MF = 0.75); and 4, 6, and 6 (SCN⁻ MF = 1.0), respectively. The continuous lines are fits with the logistic Eq. 2 to determine V_{0.5_app} and slope values. (G) Changes of reversal potential induced by SCN⁻ MF on currents generated by I641A in 0 Ca²⁺ (lime green), I641A in 0.2 µM Ca²⁺ (gray), and E702Q/E705Q in 12 µM Ca²⁺ (salmon) channels. **, ΔEr values for the I641A and E702Q/E705Q channels were statistically different (P = 0.01) from those corresponding to WT (blue line). Data were analyzed using one-way ANOVA with a Tukey’s post-hoc test. (H)P_SCN/P_Cl for I641A in 0 Ca²⁺ (lime green), I641A in 0.2 µM Ca²⁺ (gray), and E702Q/E705Q in 12 µM Ca²⁺ (salmon) channels at different SCN⁻ MF. Permeability ratios were calculated using the Goldman–Hodgkin–Katz equation and the changes of reversal potential shown in G. (I)V_{0.5_app} at different SCN⁻ MF determined for I641A in 0 Ca²⁺ (lime green), I641A in 0.2 µM Ca²⁺ (gray), and E702Q/E705Q in 12 µM Ca²⁺ (salmon) channels. Values were obtained from fits shown in B, D, and F. **, V_{0.5_app} values for the I641A channel in the presence of Ca²⁺ were statistically different (P = 0.01) from those obtained with 0 SCN⁻ MF. Data were analyzed using one-way ANOVA with a Tukey’s post-hoc test. (J) Slope values at different SCN⁻ MF determined for I641A in 0 Ca²⁺ (lime green), I641A in 0.2 µM Ca²⁺ (gray), and E702Q/E705Q in 12 µM Ca²⁺ (salmon) channels. Values were obtained from fits shown in B, D, and F.