Reproducing the cryo-EM (PDB ID 6uz0 ) pore-bound position of flecainide in the rat Na _v 1.5. (A and B) Free energy surfaces (FES, top-down and transverse views) generated from metadynamics sampling of the pore using both neutral flecainide (A) and protonated flecainide (B); FES contours are shown every 4 kJ/mol and lighter colors indicate higher energy or less favorable regions of the pore; center of mass (COM) positions of the representative pose from each of the top five clusters shown in white markers, as well as the COM position of the cryo-EM flecainide pose (cross). (C) Representative snapshot of the top cluster for neutral and protonated flecainide compared to the cryo-EM structure (showing top-down and transverse views of the pore). (D) RMSD of the top neutral and protonated clusters to the cryo-EM pose; the number of data points in each cluster shown above each box/violin. (E) Residue interactions between the neutral (blue) and protonated (red) flecainide identified within the top five clusters. (F) Representative poses of the top five clusters for neutral (left) and protonated (right) flecainide; dotted square highlights the positioning of flecainide’s trifluoromethylated arm in the DII–III fenestration. (G) The pore with resolved flecainide from the cryo-EM structure 6uz0, overlayed with the density around the drug. (H) Close-up of the flecainide cryo-EM density colored by the OccuPy scale value, where values closer to 1 represent better relative local resolution in the cryo-EM map.