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

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.

We employed site-directed mutagenesis, electrophysiological recordings and structural modelling with docking simulations to analyse flecainide interactions with Na_v1.2 (brain), Na_v1.4 (skeletal muscle) and Na_v1.5 (cardiac) isoforms in the resting, open and inactivated states.

Flecainide showed the highest affinity for Na_v1.5, followed by Na_v1.4 and the lowest for Na_v1.2 across all states. Within each isoform, affinity ranked as inactivated > open > resting. In Na_v1.5, 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.

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.