Single mutants I287D (A–C) and F324D (D–F) are insensitive to extracellular Mg²⁺. (A and D) Mg²⁺ shifts the voltage dependence of activation in (A) I287D and (D) F324D channels. Values of g/g_max obtained in the absence (■) or presence (•) of Mg²⁺ were plotted versus voltage. Data are provided as mean ± SEM; n = 6 (I287D) or 8–9 (F324D). Data were fitted with a single Boltzmann function. Values of V_1/2 and apparent valence for I287D in the absence and presence of Mg²⁺ were −20 ± 2 mV and 1.7 ± 0.1, and 0 ± 1 mV and 1.5 ± 0.2, respectively. Values of V_1/2 and apparent valence for F324D in the absence and presence of Mg²⁺ were 21 ± 1 mV and 2.1 ± 0.2, and 40 ± 1 mV and 2.5 ± 0.3, respectively. To correct for the field effect of Mg²⁺, g/g_max values obtained in Mg²⁺ were shifted by −20 mV and replotted (○). (B and E) Representative I287D (B) and F324D (E) currents recorded at +20 mV in the absence (solid trace) and +40 mV in the presence (dashed trace) of Mg²⁺ have been scaled and overlaid. Values of τ_act (I287D) or τ_act,_fast (F324D) obtained in the absence (+20 mV) and presence (+40 mV) of Mg²⁺ did not differ significantly and were 2 ± 0.1 and 2 ± 0.1 ms for I287D, and 17 ± 2 and 14 ± 1 ms for F324D, respectively. (C and F) Mg²⁺ does not affect activation kinetics in (C) I287D or (F) F324D. I287D and F324D current traces recorded in the absence (■) or presence (•) of Mg²⁺ were fitted with single- (I287D) or double- (F324D) exponential functions. Values of τ_act were plotted versus voltage. In F, only the major, fast kinetic component of F324D activation is shown. Data are shown as mean ± SEM; n = 6 (I287D) or 3 (F324D). To correct for the field effect of Mg²⁺, τ_act values obtained in Mg²⁺ were shifted by −20 mV and replotted (○).