High pH sensitivity of Kv11.1 is revealed at low Ca²⁺ and depends on the EAG-specific acidic charges. (A) Normalized GV relations for Kv11.1 are shown for pH 6, 7, and 8 at 1 mM Ca²⁺ and for pH 6 and 8 at 50 µM Ca²⁺. Conductance values were determined from isochronal tail currents recorded at −40 mV after 4-s steps to the indicated voltages from a −100-mV holding potential. Data points show mean ± SEM (n = 4–10), and curves show Boltzmann fits; V₅₀, slope factors, and ΔV₅₀ (pH 8 to 6) are reported in Table 1 and Fig. 12. (B) ΔV₅₀ (pH 8 to 6) for WT Kv11.1 is plotted as a function of Ca²⁺ concentration and fitted with a four-parameter dose–response curve. Log₁₀ scale is applied to the Ca²⁺ concentration, and data points show mean ± SEM. (C–E) Normalized GV relationships for the EAG-specific charge mutants D460C (D5C) and D509C (D6C) and the universal acidic charge mutant D456C (D1C) are shown for pH 6, 7, and 8 at 1 mM Ca²⁺ and pH 6 and 8 at 50 µM Ca²⁺. Conditions are coded by shading and color as in A. Single Boltzmann fit parameters and ΔV₅₀ (pH 8 to 6) are reported in Table 1 and Fig. 12, respectively. (F) ΔV₅₀ (pH 8 to 6) for Kv11.1 acidic neutralization mutants at 50 µM and 1 mM Ca²⁺ are presented in comparison with the Kv11.1 WT fit curve from B.