pH_i alters Ca²⁺ binding affinity of TMEM16F. (A) Representative TMEM16F currents recorded from inside-out patches perfused with intracellular solutions containing 0.1, 1, 5, 100, 1,000, and 5,000 µM Ca²⁺ at different pH_i values (5,000 µM at pH 6.1 only). (B) Ca²⁺ dose–response of mTMEM16F channel at +100 mV with different pH_i values. The smooth curves represent the fits to the Hill equation: G/G_max = G_1,000/[1 + (K_d/[Ca²⁺])H], where K_d is the apparent dissociation constant, H is the Hill coefficient, and G_1,000 is the conductance with 1,000 µM Ca²⁺ at given pH_i. The error bars represent SEM (n = 5). (C) EC₅₀ values of Ca²⁺ at pH_i 6.1, 7.3 and 8.9 were 144.45 ± 6.80, 6.20 ± 0.82, and 1.24 ± 0.14 µM, respectively. The error bars represent SEM (n = 5). P values were determined with Tukey test comparisons after one-way ANOVA: ****, P < 0.0001. (D) The G-pH_i relationship of TMEM16F current at +100 mV under different Ca²⁺ concentrations. Solid lines represent linear fits. (E) The relationship of pH_i sensitivity and [Ca²⁺]_i concentration. The pH_i sensitivity values were slopes from linear fit shown in D under different Ca²⁺ concentrations. The smooth line was fitted with a bell-shaped dose–response curve using GraphPad Prism, with peak pH sensitivity of 0.33 at ∼15 µM Ca²⁺. The error bars represent SEM (n = 5).