Mg²⁺ significantly slows activation and increases the delay before pore opening in I287D+F324D channels. (A) I287D+F324D currents were recorded in the absence (left) or presence (middle) of Mg²⁺. (Right) After approximate correction for the nonspecific field effect of Mg²⁺ (see B), current traces have been scaled and overlaid. Representative current traces were recorded at +20 mV in the absence (dashed trace) or at +30 mV in the presence (dotted trace) of Mg²⁺. Fitted exponential functions (solid lines) have been superimposed. (B) Mg²⁺ shifts the voltage dependence of activation in I287D+F324D channels. Because the conductance of I287D+F324D channels did not saturate in the tested voltage range, un-normalized conductance values from a representative experiment are shown. Data were obtained in the absence (■) or presence (•) of Mg²⁺, plotted versus voltage, and fitted with a single Boltzmann function without constraining the maximum conductance value. Fitted parameters from individual experiments were averaged and are provided as mean ± SEM; n = 8. Values of V_1/2 and apparent valence in the absence and presence of Mg²⁺ were 38 ± 2 mV and 0.7 ± 0.04, and 43 ± 1 mV and 1.0 ± 0.07, respectively. To correct for the field effect of Mg²⁺, conductance values obtained in Mg²⁺ were shifted by −5 mV and replotted (○). (C) Mg²⁺ slows activation kinetics in I287D+F324D. (Left) Values of τ_act obtained in the absence (■) or presence (•) of Mg²⁺ have been plotted versus voltage from 0 to +45 mV. To correct for the field effect of Mg²⁺, τ_act values obtained in Mg²⁺ were shifted by −5 mV and replotted (○). Data are provided as mean ± SEM; n = 7. Shifted τ_act values obtained with Mg²⁺ and τ_act values obtained without Mg²⁺ differed significantly; P < 0.05. (Right) Values of τ_act obtained at voltages from +50 to +95 mV are shown on an expanded scale; symbols as described above. To correct for the field effect of Mg²⁺, τ_act values obtained in Mg²⁺ were shifted by −5 mV and replotted (○). Shifted τ_act values obtained with Mg²⁺ and τ_act values obtained without Mg²⁺ differed significantly; P < 0.05. (D) Representative current traces recorded at +20 mV in the absence (solid trace) or at +25 mV in the presence (dashed trace) of Mg²⁺ are shown on an expanded time scale to highlight the delay before pore opening. The arrow indicates start of voltage pulse (t = 0). Values of τ_act obtained in the absence (+20 mV) and presence (+25 mV) of Mg²⁺ differed significantly and were 9.9 ± 0.3 and 20.2 ± 0.5 ms, respectively (n = 7; P < 0.05). Measured delay values were 4.6 ± 0.2 ms at +20 mV in the absence and 11.3 ± 0.8 ms at +25 mV in the presence of Mg²⁺ (n = 7; P < 0.05). (E) Mg²⁺ increases the delay before activation of the ionic conductance in I287D+F324D. The delay before pore opening was measured in the absence (■) or presence (•) of Mg²⁺ and plotted versus voltage. Data are provided as mean ± SEM; n = 10. To correct for the field effect of Mg²⁺, delays measured in the presence of Mg²⁺ were shifted by −5 mV and replotted (○). Shifted delay values obtained with Mg²⁺ and delay values obtained without Mg²⁺ differed significantly; P < 0.05. (F) Mg²⁺ does not affect deactivation kinetics. Representative tail currents evoked at −95 mV in the absence (dashed trace) or at −90 mV in the presence (dotted trace) of Mg²⁺ have been scaled and overlaid. Values of τ_deact in the absence (−95 mV) and presence (−90 mV) of Mg²⁺ were 3.7 ± 0.5 and 3.9 ± 0.1 ms, respectively, and did not differ significantly. (G) The membrane was depolarized to +40 mV, followed by repolarization for 200 ms to tail potentials ranging from −40 to −115 mV. Tail current traces were fitted with single-exponential functions to obtain values for τ_deact in the absence (■) or presence (•) of Mg²⁺, which were plotted versus repolarization voltage. Data are provided as mean ± SEM; n = 4. To correct for the field effect of Mg²⁺, τ_deact values obtained in Mg²⁺ were shifted by −5 mV and replotted (○).