Flecainide binding dynamics in voltage‐gated sodium channels: A comparative isoform analysis of the state‐dependent block

Background and Purpose Local anaesthetics and class I anti‐arrhythmic drugs block voltage‐gated Na + channels, but the molecular basis for the isoform differences in drug sensitivity remains unclear. Understanding these mechanisms is essential for optimizing therapeutic efficacy and safety. Flecainide, a class Ic anti‐arrhythmic agent, is also used for non‐dystrophic myotonias but lacks use in epilepsy. This study aimed to elucidate the mechanisms underlying the isoform differences in the state‐dependent block of flecainide. Experimental Approach We employed site‐directed mutagenesis, electrophysiological recordings and structural modelling with docking simulations to analyse flecainide interactions with Na v 1.2 (brain), Na v 1.4 (skeletal muscle) and Na v 1.5 (cardiac) isoforms in the resting, open and inactivated states. Key Results Flecainide showed the highest affinity for Na v 1.5, followed by Na v 1.4 and the lowest for Na v 1.2 across all states. Within each isoform, affinity ranked as inactivated > open > resting. In Na v 1.5, alanine substitution of a phenylalanine (Phe) residue in DIV‐S6 caused the greatest affinity reduction, particularly in the open and inactivated states. Structural models revealed isoform‐ and state‐dependent binding poses, with the piperidine nitrogen consistently pointing towards the selectivity filter and positioned higher in the open and inactivated states. Phe in DIV‐S6 contributed to high‐affinity binding, and the distance between the aromatic ring centers of flecainide and Phe correlated with the isoform differences in high affinities and the state‐dependent block within each isoform. Conclusion and Implications This study clarifies the structural basis for isoform‐ and state‐dependent drug affinities, providing insights that may guide development of isoform‐selective drugs with improved therapeutic profiles.