Regulation of ANO1 P_HCO3/P_Cl by calmodulin. (A–C) Supplementation of calmodulin in inside-out patches increases P_HCO3/P_Cl of hANO1. Cytosolic solution includes 3 µM of free Ca²⁺. (A) The addition of human calmodulin altered bi-ionic potential of Cl⁻/HCO₃⁻ and increased P_HCO3/P_Cl of hANO1 from 0.27 to 0.64. (B) I-V relationships were analyzed during the zero-current clamp recording in A. The anion outward chord conductance (G) between E_rev and E_rev plus 25 mV was calculated by linear plotting. (C) Effect of the recombinant calmodulin (His₆-tagged bovine calmodulin) was much less than that of human calmodulin. (D and E) Bi-ionic potentials as a function of membrane conductance are plotted. The hANO1 membrane potential data from zero-current clamping in whole-cell configuration were obtained from our previous study (Jung et al., 2013). The bath solution was replaced from 150 mM Cl⁻ to 130 mM HCO₃⁻ plus 20 mM Cl⁻ (D) or to 130 mM I⁻ plus 20 mM Cl⁻ (E). Currents were activated by 400 nM (black) or 3 µM of free Ca²⁺ (red) in the pipette. The linear lines represent the result of regression analyses. Large current amplitude did not produce a significant voltage drop. (F) Effect of the CBD mutation (I317A + I762A) on the Cl⁻ conductance of hANO1. Whole-cell currents were activated by 3 µM of free Ca²⁺ in the pipette. Cl⁻ membrane conductance was obtained by voltage clamping at 40 mV, and the values were normalized by cell capacity. The normalized conductance of wild-type and CBD-mutated ANO1s were 8.19 ± 0.84 (n = 5) and 8.38 ± 0.87 (n = 10) nS/pF, respectively. Data are means ± SEM. There were no significant differences between two groups (P > 0.05). The electrophysiological recordings were performed as described previously (Jung et al., 2013).