Shaker-IR is insensitive to extracellular Mg²⁺. (A) Shaker-IR currents were recorded in the absence (left) or presence (middle) of 10 mM of extracellular Mg²⁺. From a holding potential of −100 mV, 50-ms test pulses to voltages between −60 and +90 mV were applied in 10-mV increments. In this and subsequent figures, traces obtained by pulses ranging from −60 to +40 mV are shown. (Right) Currents recorded at −10 mV in the absence (solid trace) or presence (dashed) of Mg²⁺ have been scaled and overlaid. (B) Mg²⁺ shifts the voltage dependence of activation in Shaker-IR channels. Conductance values were calculated from steady-state current amplitudes during a 50-ms test pulse. Normalized conductance values (g/g_max) obtained in the absence (■) or presence (•) of Mg²⁺ were plotted versus voltage. Datasets were fitted with single Boltzmann functions. Values of V_1/2 in the absence and presence of Mg²⁺ were −19 ± 1 and −7 ± 2 mV, respectively. Values of the apparent valence were 2.5 ± 0.2 and 2.5 ± 0.1 without and with Mg²⁺, respectively. To correct for the field effect of Mg²⁺, g/g_max values obtained in Mg²⁺ were shifted by −12 mV and replotted (○). Data are provided as mean ± SEM; n = 6. (C) Current traces have been scaled and overlaid after approximate correction for the surface charge effect of Mg²⁺. 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. Values of τ_act in the absence (+20 mV) or presence (+30 mV) of Mg²⁺ were 1.2 ± 0.1 and 1.1 ± 0.1 ms, respectively, and did not differ significantly (P ≥ 0.05). (D) Activation kinetics are unaffected by Mg²⁺. τ_act in the absence (■) or presence (•) of Mg²⁺ were obtained by fitting single-exponential functions to the current traces and plotted versus voltage. To correct for the field effect of Mg²⁺, τ_act values obtained in Mg²⁺ were shifted by −12 mV and replotted (○). Data are shown as mean ± SEM; n = 6. (E) Tail current traces have been scaled and overlaid after approximate correction for the surface charge effect of Mg²⁺. Representative tail currents were recorded at −100 mV in the absence (dashed trace) or at −90 mV in the presence (dotted trace) of Mg²⁺. Fitted exponential functions (solid lines) have been superimposed. The values of τ_deact in the absence (−100 mV) and presence (−90 mV) of Mg²⁺ did not differ significantly and were 1.4 ± 0.1 and 1.5 ± 0.1 ms, respectively. (F) Mg²⁺ does not affect deactivation kinetics. The membrane was depolarized to +40 mV, followed by repolarization to tail potentials ranging from −50 to −125 mV. Tail currents recorded in the absence (■) or presence (•) of Mg²⁺ were fitted with a single-exponential component to obtain values for τ_deact, which were plotted versus repolarization voltage. Data are provided as mean ± SEM; n = 9. To correct for the field effect of Mg²⁺, values of τ_deact obtained in the presence of Mg²⁺ were shifted by −12 mV and replotted (○).