The Ca ²⁺ -pocket of site 1 regulates anion selectivity in TMEM16A. (A) Six sets of I-V relationships color-coded according to the extracellular solution’s anion (top center). Black, Cl⁻; olive, Br⁻; red, NO₃⁻; cyan, I⁻; and navy, SCN⁻. We used these relationships to determine the reversal potentials of the currents through WT, E702Q, E705Q, E702Q/E705Q, and 5Q mutant channels. Currents were recorded from cells dialyzed with an internal solution containing 0 (5Q), 0.2 (WT), or 12 (E702Q, E705Q, E702Q/E705Q, and 5Q) µM Ca²⁺. (B) Comparison of the anion permeability ratios for WT TMEM16A (gray), E702Q (pink), E705Q (orange), E702Q/E705Q (blue), 5Q without Ca²⁺ (lime green), and 5Q in the presence of Ca²⁺ (sea green). Permeability ratios were 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: [X⁻]_e/[Cl⁻]_i = 140/40 mM and pH_e = pH_i = 7.3. **, P_X/P_Cl values were statistically different (P = 0.01) from those corresponding to the WT channel. The data were statistically analyzed using one-way ANOVA with a Tukey’s post-hoc test.