Architecture of HCN4 channel pore and filter binding sites for K⁺ ions. (A) Cross-section of the HCN4 PD in cartoon representation (PDB ID 7NP3). For clarity, only two opposing subunits of the homotetramer and ions K⁺ bound to the SF are shown. The main channel axis, centered in the pore and perpendicular to the membrane surface, is outlined as a z-vector pointing from the extracellular side (ext.) toward the cytosol (cyt.). (B) Schematic representation of the HCN4 SF. Carbonyl oxygen planes constituting the SF are marked as p23 (I480-CO) and p34 (I479-CO), and are indicated as black dashed lines. The three main binding sites for K⁺ are marked as Sa, Sb, and Sc; S3 refers to the respective K⁺ binding site in canonical K⁺ channels (Zhou et al., 2001). (C) Data here shows comparison of the selectivity filter of the canonical channel KcsA (PDB ID 1K4C). The pore region above and below the SF are termed as vestibule (ves.) and cavity (cav), respectively. (D) Detailed depiction of K⁺ conductance described in previous MD studies (Saponaro et al., 2021a). The protein backbone of SF residues is depicted in stick representation in all subplots, with hydrogen, carbon, nitrogen, and oxygen atoms colored in light gray, gray, blue, and red, respectively.