Extracellular Zn²⁺ delays pore opening in I287H+F324H channels. (A) Representative current traces, evoked by depolarizing from −80 to +40 mV in the absence (black) or presence (red) of 1 µM Zn²⁺, are shown on an expanded scale to illustrate the effect of Zn²⁺ on the delay before pore opening. Delays were determined by extrapolating fitted single-exponential functions (dashed green lines) to the zero current level (dashed gray line) (Perozo et al., 1994; Lin et al., 2010). (B) The delay before pore opening was measured at test voltages ranging from +10 to +80 mV in the absence (black squares) or presence (red circles) of 1 µM Zn²⁺. Delay values differed significantly in the presence and absence of Zn²⁺, as indicated (*, P < 0.05; ‡, P < 0.005). Values are shown as mean ± SEM (n = 4–10). At +40 mV, delays measured with and without Zn²⁺ were 3.4 ± 0.6 ms and 0.6 ± 0.2 ms, respectively. (C) Zn²⁺ shifts the dependence of the delay on prepulse potential in the depolarized direction. Delay values have been plotted on an inverted scale as a function of prepulse potential. Delays were measured in the absence (black squares) or presence (red circles) of 1 µM Zn²⁺ by stepping from the holding potential of −80 mV to prepulse voltages ranging from −130 to 25 mV for 20 ms before depolarizing to +60 mV. Delay data were fitted with single Boltzmann functions (solid lines). Values of V_1/2 and slope were −39 ± 1 mV and 18 ± 1 mV, and −14 ± 3 mV and 12 ± 2 mV, respectively, in the absence and presence of Zn²⁺ (n = 6–8). Normalized conductance values, measured as a function of test potential in the absence (open black squares) or presence (open red circles) of Zn²⁺, are shown on the same axes. Conductance data fitted with single Boltzmann functions (solid lines) with (red) or without (black) Zn²⁺ did not differ significantly. Values of V_1/2 and slope were 44 ± 1 mV and 17 ± 1 mV, and 43 ± 1 mV and 18 ± 1 mV, respectively, in the absence and presence of Zn²⁺ (n = 5). For clarity, error bars have been omitted from the figure.