BPS2026 – Modulation of the cardiac sodium channel NaV1.5 by the sea anemone peptide anthopleurin A (ApA)

Voltage-gated sodium (NaV) channels are essential mediators of excitability in muscle and nerve cells. The cardiac isoform, NaV1.5, drives the rapid depolarization phase of the cardiac action potential, and its dysfunction is directly linked to inherited arrhythmia syndromes such as long QT and Brugada. While sea anemone toxins are known to alter NaV channel gating, the structural determinants of their activity at NaV1.5 have not been defined. Here, we report an integrated structural and functional analysis of the NaV1.5 DIV S3-S4 motif and its interaction with sea anemone toxin, Anthopleurin A (ApA). Using nuclear magnetic resonance (NMR) spectroscopy in dodecylphosphocholine micelles, we characterized the structural and dynamic properties of the isolated NaV1.5 DIV S3-S4 motif in the absence and presence of ApA toxin. These studies identified residues I1612 and S1618 as important contributors to toxin binding. To test their functional significance, we generated alanine substitutions at these sites and assessed channel activity in the presence of ApA using whole-cell patch clamp electrophysiology. Mutations at both positions significantly altered toxin sensitivity, confirming their role in toxin-paddle interactions. Current work on identifying additional residues in toxin binding through NMR and electrophysiology experiments is ongoing and will allow painting a more complete structural picture of toxin selectivity and mode of action. These findings will contribute to the development of future peptide-based strategies to fine-tune cardiac sodium channel activity.