Cation-dependent modulation of distances between filter carbonyl oxygens. (A–D) Inter carbonyl-oxygen distance histograms and overlayed KDE plots for different cation species in p23 are depicted for (A) subunits A|C and (B) B|D, distances between these subunits in p34 are shown in C and D. Reference distances derived from the cryoEM structure are depicted as black dashed lines. Distances were extracted from simulations conducted between −150 and −700 mV for the HCN4 pore domain. Only distances associated with binding events lasting ≥12 ns are considered. Counts in histogram bins were normalized to yield a total histogram area equal to 1. (E and F) Representative frames for the largest and smallest cations bound and associated carbonyl oxygen rearrangements are shown for (E) p23 with Na⁺ and Cs⁺ and (F) p34 with Li⁺ and Rb⁺. Here, carbonyl groups in p23/p34 are shown as stick representation, with red spheres at the position of carbonyl oxygens in the widened state (i.e., with Cs⁺ and Rb⁺ proximate to p23 and p34, respectively). Gray spheres indicate the carbonyl oxygen position for states with Na⁺ and Li⁺ in p23 and p34, respectively. Carbonyl oxygen planes are indicated by solid lines, and ∆p23 and ∆p23 values for both subunits are indicated by dashed lines with cyan for Na⁺, green for Li⁺, red for Rb⁺, and orange for Cs⁺.