R294H-A351H forms a high affinity Zn²⁺ binding site that reduces current magnitude. (A) Representative current traces from an oocyte expressing Kv1.2-R294H-A351H channels recorded in the absence (left) and presence (right) of 1 μM Zn²⁺, using the protocol outlined in the Materials and methods. Black arrowheads indicate where steady-state current was measured to generate I-V and G-V relationships. (B) Mean current–voltage relationships from oocytes expressing Kv1.2-R294H-A351H channels recorded in the absence (solid squares, n = 8) and presence (open circles, n = 8) of 1 μM Zn²⁺. (C) Normalized G-V curves for Kv1.2-R294H-A351H obtained in the absence (black squares, n = 8) and presence of various concentrations of Zn²⁺; 10 nM (gray triangles, n = 7), 100 nM (open diamonds, n = 7), and 1 μM (open circles, n = 8) showing dose-dependent reduction in conductance with increasing concentrations of extracellular Zn²⁺. To demonstrate loss of current with Zn²⁺ application, conductance (G) values obtained in either the absence or presence of Zn²⁺ were normalized to the maximal G value obtained in the absence of Zn²⁺. Data for control (no Zn²⁺) and 10 nM Zn²⁺ were fitted with a single Boltzmann function, shown as solid black and gray curves respectively. (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 (n = 9) and (E) Kv1.2-A351H (n = 8). Single mutants showed no loss of current magnitude with application of 1 μM Zn²⁺; hence G values were normalized to the maximal G in each experimental condition (absence or presence of Zn²⁺).