SPM-binding kinetics. (A) Voltage-dependent SPM binding times (Sims. 31, 32, 35, 36, 39–41, and 43–45), with binding defined as when the distance from SPM to the D173 center of mass (C_α atoms) was 10 Å or less (30-ns running medians). (B) SPM binding (at 310 mV) to the CTD in the E225A/E300A CTD double mutant (Sims. 91 and 92), in WT Kir2.2 (Sims. 31 and 32), and in ROMK (Sims. 153 and 154). Binding was defined as when the distance from SPM to the E225/E300 center of mass (C_α atoms) was 10 Å or less (30-ns running medians). (C) SPM snapshots (cyan) during binding (Sim. 35) to the extended pore spanning the entire protein (gray cut-through surface). SPM center of mass number density along the (extended) pore-axis in WT with two or four rectification controller (D173) residues deprotonated, and in the E225A/E300A CTD double mutant. (D) Ion permeation events per unit time in the presence and absence of SPM (Sims. 36–46, red; Sims. 9–11, black) at different depolarizing voltages. (E) Voltage-dependent SPM binding (Fig. S4 A) and SPM residence times at the pore cavity-SF region with two and with four D173 residues deprotonated (Sims. 31–42, 150–152).