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Figure 5. E283 is not required for Mg2+ modulation of activation time course in I287D+F324D. (A) E283A+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), representative current traces recorded at +20 mV in the absence (dashed trace) or at +30 mV in the presence (dotted trace) of Mg2+ have been scaled and overlaid. Single-exponential fits to the data are shown (solid curves). (B) Mg2+ shifts the voltage dependence of activation in E283A+I287D+F324D. Because the conductance of E283A+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 V1/2 and apparent valence in the absence and presence of Mg2+ were 36 ± 3 mV and 0.8 ± 0.07, and 44 ± 4 mV and 0.9 ± 0.07, respectively. To correct for the field effect of Mg2+, conductance values obtained in Mg2+ were shifted by −8 mV and replotted (○). (C) Mg2+ slows activation kinetics. (Left) Values of τact obtained in the absence (■) or presence (•) of Mg2+ have been plotted versus voltage from 0 to +48 mV. Data are provided as mean ± SEM; n = 8. To correct for the field effect of Mg2+, τact values obtained in Mg2+ were shifted by −8 mV and replotted (○). Shifted τact values obtained with Mg2+ and τact values obtained without Mg2+ differed significantly; P < 0.05. (Right) Values of τact obtained at voltages from +50 to +98 mV are shown on an expanded scale; symbols as described above. To correct for the field effect of Mg2+, τact values obtained in Mg2+ were shifted by −8 mV and replotted (○). Shifted τact values obtained with Mg2+ and τact values obtained without Mg2+ differed significantly; P < 0.05. (D) Representative current traces recorded at +20 mV in the absence (solid trace) or at +28 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 τact measured in the absence (+20 mV) and presence (+28 mV) of Mg2+ were 11.3 ± 0.5 and 18.5 ± 1.2 ms, respectively, and differed significantly (P < 0.05; n = 8). Measured delay values were 3.3 ± 0.3 ms at +20 mV in the absence and 9.1 ± 0.2 ms at +28 mV in the presence of Mg2+ and differed significantly (P < 0.05; n = 8). (E) Mg2+ increases the delay before activation of the ionic conductance in E283A+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 −8 mV and replotted (○). Shifted delay values obtained with Mg2+ and delay values obtained without Mg2+ differed significantly; P < 0.05. Image published in: Lin MC et al. (2010) © 2010 Lin et al. Creative Commons Attribution-NonCommercial-ShareAlike license Larger Image Printer Friendly View

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