Extracellular Zn²⁺ slows activation in I287H+F324H channels. (A) Currents were evoked by depolarizing from −80 to +60 mV in the absence (black) or presence (red) of 1 µM Zn²⁺. Traces were fitted with single-exponential functions (dashed green lines) to provide activation time constants (τ_act). (B) τ_act values measured at test voltages ranging from +10 to +90 mV in the absence (black squares) or presence (red circles) of 1 µM Zn²⁺ differed significantly (§, P < 0.0005) at all voltages. Data are shown as mean ± SEM (n = 13). At +60 mV, τ_act values were 66 ± 4 ms and 8.6 ± 0.6 ms with and without 1 µM Zn²⁺, respectively. (C) Values of τ_act at +60 mV were plotted versus Zn²⁺ concentration and fitted with a rectangular hyperbola (black line) to determine [Zn²⁺]_1/2, which was 0.12 µM (n = 6–13). (D) The box plot shows τ_act values measured at +60 mV in the indicated concentrations of Zn²⁺ for I287H+F324H, the Shaker-IR parent channel, and the I287H and F324H single-mutant channels. Mean values of τ_act that differed significantly from no Zn²⁺ are indicated: *, P < 0.05; ‡, P < 0.005; §, P < 0.0005 (n = 2–13).