SF substates and the movement of the ions through the conduction pathway of KCNQ1. (A) Definition of the SF substates used in this study based on the distance of permeating K⁺ ions from the center of mass of four carbonyl carbons of G314 residues. Frequently observed occupancy substates of the SF are shown above the diagram. Scav, K⁺ binding site in the cavity. (B) Sample of diagrams showing K⁺ movement through the SF of the channel at two different K⁺_o concentrations investigated. Definitions and symbols are the same as in A. S denotes the spontaneous release of K⁺, C denotes the permeations according to the canonical mechanism (K⁺_in-induced release). Each colored line represents the movement trajectory of one K⁺ ion. Ion trajectories in the internal medium are removed for clarity. (C) Summary of the SF substate distribution at two different concentrations of K⁺_o as indicated. (D) Statistical analyses of probability of key SF substate occupancies at 5 and 150 mM K⁺_o conditions. Error bars in the graphs represent ± SEM; S3-S2 (P = 0.00121); S_cav-S₃-S₂ (P = 0.0013); S_cav-S₃-S₂ (P = 0.00217); n.s., not significant. Student’s unpaired t test was used to compare the groups.