The TM6 hinge and hydrophobic gate regulate TMEM16A anion selectivity. (A) Example of I-V relationships used to determine the reversal potentials of the currents through the G644P channel. Currents were recorded from a cell exposed to the indicated anions (top left) and dialyzed with an internal solution containing 0.2 µM Ca²⁺. (B) Comparison of the anion permeability ratios in WT TMEM16A (gray, from Fig. 6) and G644P (green) channels. Permeability ratios were calculated from changes of reversal potentials induced by the total replacement of extracellular Cl⁻ as shown in A. The permeability ratios were calculated under the following conditions: [X⁻]_e/[Cl⁻]_i = 140/40 mM and pH_e = pH_i = 7.3. (C) Two sets of I-V relationships color-coded according to the extracellular solution’s anion. Black, Cl⁻; olive, Br⁻; red, NO₃⁻; cyan, I⁻; and navy, SCN⁻. The currents were recorded from two cells expressing WT or I641A channels. WT channels were recorded in the presence of 0.2 μM Ca²⁺, while I641A in 0.2 μM Ca²⁺ and 0 Ca²⁺. For clarity, only the currents around the reversal potentials are shown, and those were fitted with a polynomial function to determine the reversal potentials by interpolating on the voltage axis. (D) Anion permeability ratios for WT and I641A channels calculated from changes of reversal potential induced by the total replacement of extracellular Cl⁻ with the indicated anions. The permeability ratios were calculated under the following conditions: 0 (I641A) and 0.2 (WT and I641A) µM Ca²⁺, [X⁻]_e/[Cl⁻]_i = 140/40 mM and pH_e = pH_i = 7.3. **, P_X/P_Cl values for the I641A channel at 0 or 0.2 µM Ca²⁺ were statistically different (P = 0.01) from the WT channels. The data were statistically analyzed using one-way ANOVA with a Tukey’s post-hoc test.