Characteristic ion trajectories depicting primary binding sites of Li ⁺ , Rb ⁺ , and Cs ⁺ obtained from simulations conducted at −700 mV in solutions with a single cation species. (A) With prolonged presence of Li⁺, p34 was identified as a single binding site within the SF. Permeation of Li⁺ through p23 was only observed in a single instance. (B) Similar to K⁺, Sa (encompassing p23) and Sb/Sc (proximate to p34) were identified as binding sites within the SF for Rb⁺. Intermediate fluctuations between Sb and S3 in the single-ion state are observable at higher voltages, while prolonged binding to S3 occurred at lower voltages. (C) Cs⁺ is predominantly found as a single cation in the S3 site in all simulations. Both Rb⁺ as well as Cs⁺ are found in a {Sa, Sc} configuration during conduction events. (D) Empirical CDF of computed numerical densities (in counts/nm³) for Li⁺, Rb⁺, and Cs⁺ conducted at −700 mV (n = 4, n = 1, and n = 2, respectively), including simulations depicted in A–C and of K⁺ (n = 4; pure KCl), and Rb⁺ (n = 3, pure RbCl) at −500 mV along the z-axis of the simulation box. The mean z positions of p23, p34, and the POPC-headgroup phosphor atoms (Phos.) are marked as dashed lines. Arrowheads denote reference z_rel positions along the pore axis: Unequal count densities are observed for Li⁺ compared to the other cations (I.), whereas similar number densities are observed in the vestibule above the SF (II.). For reference, the channel pore-facing helix and the SF of a single HCN4 monomer are shown above the subplot. The vestibule (ves.) and SF binding sites Sa, S3, and Sb are separately outlined.