Cu²⁺ action is independent of native Cys and His residues but is pH dependent. (A) G_K-V relations for WT (○: 0 Cu²⁺; •: 100 Cu²⁺) and C14A/C141A/C277A (“C3A”, ▵: 0 Cu²⁺; ▴: 100 Cu²⁺) show similar inhibition by 100 μM Cu²⁺. (B) G_K-V relations for WT (○: 0 Cu²⁺; •: 100 Cu²⁺) and H254A (<: 0 Cu²⁺; ▾: 100 Cu²⁺) also respond similarly to 100 μM Cu²⁺ as indicated by the shift in V_0.5. (C) Mean dose–response relations (G_K-[Cu]²⁺) measured at voltages near V_0.5 for WT (○), C14A/C141A/C277A (▵), and H254A (▾) are indistinguishable. The solid line is a fit by the Hill equation (IC₅₀ = 2.0 μM, n_H = 0.98) (D) Dependence of Cu²⁺ action on extracellular pH (pH_O) is fit by Hill equation (pH_0.5 = 6.0, n_H = 1.15). G_K-pH_O relation for WT channels at +220 mV in 0 Ca²⁺ represents G_K in 100 μM Cu²⁺ normalized by G_K in 0 Cu²⁺ at each pH_O.