Structure of sodium channel pore module and pore-blocker sampling method. (A) Topology of sodium channel transmembrane helices (S1–S6) and the re-entrant selectivity filter (SF) loop arranged in four pseudo-tetrameric domains (DI–DIV); conserved S6 residues that form the pore binding site are labeled—rat Na_v1.5 numbering. (B) Components of the simulations featuring a drug molecule (blue stick representation) inside the closed sodium channel pore (shown as a cut-through side view with two of the domains visible), embedded in POPC bilayer (sphere representation) and solvated in NaCl ions; dotted blue line indicates the region sampled by the drug in enhanced simulations and red cross represents the drug’s center of mass (COM) and red dotted lines define the central pore axis and the drug COM relative to the central axis. (C) The pore module is formed by the S5 and S6 helices and the selectivity filter (SF), shown in new cartoon representation; positions of the conserved drug binding residues shown in stick representation. (D) Top-down view of a cut-through representation of the pore module showing the regions of the central cavity and four orthogonal fenestrations between the four domains that were sampled by drugs; red dashed lines depict the angle used to define the polar coordinates of the drug COM (red cross).