Metal ion binding to R294H-A351H stabilizes the activated state after neutralization of acidic residues D352 and E353. (A) Representative current traces from an oocyte expressing Kv1.2-R294H-A351H-D352G-E353S channels recorded in the absence (left) and presence (right) of 1 μM Zn²⁺, using the protocol outlined in the Materials and methods. (B) Normalized G-V relationships from oocytes expressing Kv1.2-R294H-A351H-D352G-E353S channels recorded in the absence (solid squares, n = 9) and presence (open circles, n = 9) of 1 μM Zn²⁺. (C) A plot of ΔV_1/2 versus Zn²⁺ concentration in oocytes expressing Kv1.2-R294H-A351H-D352G-E353S channels, where ΔV_1/2 is the difference between the fitted V_1/2 values obtained in the absence and presence of varying concentrations of Zn²⁺. The data were fitted with a hyperbolic function (solid curve) with a half-maximal concentration of 0.36 ± 0.18 μM (n = 4–8). (D and E) Normalized G-V curves obtained in the absence (solid squares) and presence (open circles) of 1 μM Zn²⁺ for (D) Kv1.2-R294H-D352G-E353S (n = 7) and (E) Kv1.2-A351H-D352G-E353S (n = 7).