A subset of I287H+R1H voltage sensors binds Zn²⁺ at −80 mV. (A) The box plot shows τ_act values measured at +60 mV in the indicated concentrations of Zn²⁺ for I287H+R1H and the R1H single-mutant channel. I287H+R1H current traces were fitted with a single-exponential function in the absence of Zn²⁺ (black box, τ_act) and the sum of two exponential functions in the presence of Zn²⁺ (blue boxes, τ_fast; red boxes, τ_slow). R1H current traces were fitted with a single-exponential function in the presence and absence of Zn²⁺ (black boxes, τ_act). Mean values of τ_act, τ_fast, or τ_slow that differed significantly from no Zn²⁺ are indicated: *, P < 0.05; †, P < 0.01; ‡, P < 0.005; §, P < 0.0005 (n = 3–13). (B) The normalized amplitude of the slow component of activation measured at +60 mV in I287H+R1H channels has been plotted versus Zn²⁺ concentration (n = 4–7). The data were fitted with a rectangular hyperbola (black line) to obtain values for [Zn²⁺]_1/2 and A_slow,max, which were 9.4 µM and 0.56, respectively. (C) A_slow,max was measured as a function of prepulse voltage in the presence of a saturating Zn²⁺ concentration (200 µM; n = 4–7). The membrane was stepped from −80 mV to prepulse voltages ranging from −140 to −40 mV for 1 s before depolarizing to +80 mV.