Modification of SF ion occupancy states by external K ⁺ . (A) Cumulative outward ionic flux at low (5 mM) and high (150 mM) K⁺_o conditions. 10 simulations for each K⁺_o condition is shown. (B) Current amplitudes calculated from the trajectories shown in A. Each point corresponds to the mean current of one simulation. Student’s unpaired t test was used to compare the groups. Lines show the median of each group. (C) Schematic illustration of canonical (black arrows) and spontaneous-S₀ release (gray arrows) mechanisms. The key substates of the KCNQ1 SF are shown as snapshots from one representative trajectory at 5 mM K⁺_o (trajectory #1). The double-headed arrow denotes the fluctuation between S_cav-S₃-S₂ and S₄-S₃-S₁ substates (see also Fig. S8). (D) Occupation probability of the S₀ site by (1) K⁺ coming from the SF and (2) by external K⁺ (left). Events of K⁺_o binding to the S₀ site of the SF lasting longer than 0.4 nS are counted. (E) Distribution of K⁺ permeation events between canonical and spontaneous–S₀ modes at two different K⁺_o conditions. Average of 5 µs (10 simulations) for each concentration is shown. (F) Mean K⁺ density in the SF during MD simulations. The center of the mass of G314 Cα atoms is taken as a 0 Z coordinate. S_cav, K⁺ binding site in the channel cavity. Error bars in the graphs represent ±SEM, *P < 0.05, **P < 0.01, n.s., not significant. Student’s unpaired t test was used to compare the groups.