Permeation barrier at the pore equator. (a) The initial membrane-embedded system set up for PDB IDs 7QKO and 7QL6 structures using the color rendering schemes in Fig. 1 b. Water and ions are not shown for clarity. (b) PMF profiles for permeation of Na⁺ ion through WT closed (7QKO), WT open (7QL6), and channel pores having mutations in β (βL9′S), α (α₂L9′S), and βδε-subunits (βδεL9′S; Fig. S4 b). The positions of the key amino acids, dashed lines. (c) Water occupancy in the pore in the closed state (7QKO). Red spheres are water molecules (O atom). The side chains at 9′ and 16′ positions in the β subunit are cyan. (d) Time-averaged water density for WT and mutant channels along the pore calculated within a cylinder of radius 5 Å along the pore axis.