Gating current and gating pore measurements for R3C from a holding potential of 0 mV. (A) Gating currents induced by hyperpolarizations from a holding potential of 0 mV. Steps were increased in 10-mV increments. (B) Gating pore currents elicited by holding the cell at 0 mV and applying hyperpolarizing pulses in 10-mV increments with no leak subtraction. I_gp was measured at the end of the pulse. (C) The correlation of charge movement (Q-V: V_1/2 = −129.6 ± 3.47 mV, k = 15.7 ± 0.6 mV; n = 5) and gating pore conductance (G_gp-V: V_1/2 = −140.9 ± 4.2 mV, k = 7.2 ± 0.28 mV; n = 6) of NaChBac-R3C. Gating pore voltage dependence and gating charge voltage dependence track each other. Error bars represent SEM. (D) Cartoon illustrating our interpretation of the results. At 0 mV, R3C is located in the gating pore (left). Immediately upon hyperpolarization to −180 mV, inward gating pore current is increased by the increase in driving force for inward ion movement (middle). During the hyperpolarization to −180 mV, the S4 segment slowly translates inward, moving R2 into the critical point of the gating pore and terminating gating pore current (right).