Comparison of critical parameters from simulations with entire HCN4 proteins and isolated channel pore. (A–D, F, and G) In simulations with entire channel protein in 150 mM K⁺ (Bauer, 2021), potassium ion transitions can be observed in the apo-open (A) but not in the holo-closed form (B). Pore hydration in the cavity (C and D) and critical helix distances (F and G) at the entrance to the cavity from these simulations were taken as reference for the open and closed state of the channel, respectively. Both parameters were compared with the respective values from simulations with the isolated pore under various experimental conditions. Data in C show hydration as the number of water molecules (N_H2O) in the cavity of the entire channel protein in the apo-open (white) and holo-closed (gray) state as in A and B compared with simulation results with the isolated pore at different voltages (blue shades). Data in D show the analysis of the same parameter as in C from simulations with the isolated pore in pure or mixed cation solutions depicted on the left. Dotted and dashed lines respectively indicate mean values from the simulation of the entire protein in the open and closed states as a reference in both C and D. (E–G) Distances between C_α atoms of I511 and T515 in opposite subunits (E) depicted in F, are critical markers for the open or closed conformation of the channel. The distributions of the respective distances from simulations of the entire protein in apo-open or holo-closed form as well as the respective distances from simulations with the isolated pore run at different voltages are shown for I511 (F) and T515 (G). All simulations with the isolated open pore confirm that it remains under different simulations conditions in the open state. Only trajectory frames corresponding to t_sim ≥ 50 ns were included in these analyses.