Extracellular Zn²⁺ slows activation and delays pore opening in I287H+A359H channels. (A) At subsaturating concentrations, Zn²⁺ induced a slow component of activation in I287H+A359H channels. The membrane was depolarized from −80 to +60 mV for 2 s. Representative current traces recorded in the presence or absence of Zn²⁺ (black, 0 µM; red, 1 µM; green, 10 µM) were scaled to the same amplitude, overlaid, and fitted with one (no Zn²⁺) or the sum of two (+Zn²⁺) exponential functions (black lines). (B) The box plot shows τ_act values measured at +60 mV in the indicated concentrations of Zn²⁺ for I287H+A359H and the A359H single-mutant channel. I287H+A359H current traces were fitted with the sum of two exponential functions (blue boxes, τ_fast; red boxes, τ_slow), except at 50 µM Zn²⁺, where one component was sufficient (black box, τ_act). A359H current traces were fitted with a single-exponential function (black boxes, τ_act). Mean values of τ_act, τ_fast, or τ_slow that differed significantly from no Zn²⁺ are indicated: ‡, P < 0.005; §, P < 0.0005 (n = 3–25). (C) The normalized amplitude of the slow component of activation measured at +60 mV has been plotted versus Zn²⁺ concentration (n = 3–10). The data were fitted with a rectangular hyperbola (black line) to obtain values for [Zn²⁺]_1/2 and A_slow,max, which were 1.2 µM and 1.0, respectively. (D) Zn²⁺ increases the delay before pore opening. Representative current traces, evoked at +60 mV in the absence (black) or presence (red) of a saturating concentration (50 µM) of Zn²⁺, were fitted with single-exponential functions (green). The fitted functions were extrapolated to the zero current level (dashed line) to estimate the delay (Perozo et al., 1994; Lin et al., 2010). (E) The box plot shows the delay before pore opening measured in the absence (black box) or presence (red box) of 50 µM Zn²⁺. Mean values obtained at +60 mV were 496 ± 50 ms and 0.8 ± 0.1 ms, with and without Zn²⁺, respectively, and differed significantly (§, P < 0.0005; n = 5–9).