Voltage dependence of gating charge movement and gating pore opening of R1C measured from a holding potential of 0 mV. (A) Example of gating currents measured in a cell expressing R1C in response to hyperpolarizations from a holding potential of 0 mV. (B) Currents elicited in a cell expressing R1C by a series of hyperpolarizing pulses in 10-mV steps from a holding potential of 0 mV. No leak subtraction protocol was used. I_gp is the nonlinear current elicited by the hyperpolarizations and was measured at the end of the hyperpolarizing pulse. (C) The correlation of charge movement (Q-V: V_1/2 = −108 ± 6.4 mV, k = 23.84 ± 1.5 mV; n = 6) and gating pore conductance (G_gp: V_1/2 = −144.4 ± 4.1 mV, k = −21 ± 1.65 mV; n = 8) for NaChBac R1C. Gating pore conductance was determined by differentiating the I_gp versus voltage relationship (see Materials and methods). (D) A cartoon illustrating our interpretation of the results for R1C. R3 occupies and seals the gating pore at 0 mV. By hyperpolarizing to more negative potentials, S4 moves inward, bringing R1C into the critical position in the gating pore negative to approximately −80 mV.