Figure 3.

Effects of Mg²⁺ on gating current were measured in the absence of permeant ions and presence of extracellular tetraethyl ammonium (TEA) to block the pore. (A) I_g evoked by 0.5-ms pulses to +200 mV in the presence (dotted traces) or absence (solid traces) of Mg²⁺, from two different patches testing the effects 10 or 100 mM Mg²⁺. (B) Mean Q_C-V relations in 0 (•), 10 (▵), and 100 (♦) mM Mg²⁺ are fit by Boltzmann functions with identical equivalent charge (z_J = 0.58e) (Horrigan and Aldrich, 2002) and were normalized by the fit amplitude (Q_cMax). Q_C represents the charge distribution for closed channels, determined by fitting the initial 50–100-μs decay of I_gON with an exponential function with time constant τ_gFast and integrating the area under the rising phase and fit (Horrigan and Aldrich, 1999). (C) τ_gFast-V relations for the patch in A are shifted by −21 mV in 10 mM Mg²⁺. Relations are fit by functions of the form τ_gFast = [α(V) + β(V)]⁻¹ where α and β are forward and backward rate constants for voltage sensor activation (0 Mg: α(0) = 58600 s⁻¹, β(0) = 1930 s⁻¹; 10 Mg: α(0) = 45400 s⁻¹, β(0) = 2350 s⁻¹) (z_α = 0.3 e, z_β = −0.2e). Representative C_g-V relations obtained with admittance analysis for (D) WT or (E) E374A/E399N channels in the presence (black curves) and absence (gray curves) of 10 or 100 mM Mg²⁺ are fit by the derivative of a Boltzmann function with respect to voltage (dotted curves) with identical amplitude and charge (z_J = 0.58e). Shifts along the voltage axis indicate the change in V_hC (ΔV_hC). Arrows indicate the upper voltage limit of the fit that was determined over a range where most channels are closed (P_o < 0.1). (F) ΔV_hC = [V_hC(Mg²⁺) − V_hC(0 Mg²⁺)] for WT (♦, n = 6) and E374A/E399N (⋄, n = 4) determined from C_g-V relations.