Diverse binding poses of agonistic neurotoxins on human Nav1.6

Nav channels are key targets for various venomous toxins. Deciphering their binding poses and mechanisms of action (MOAs) will facilitate therapeutic development targeting Nav channels. Here we present distinct binding poses of three agonistic peptide toxins on human Nav1.6 through high-resolution cryo-EM structures. The globular β-scorpion toxin Cn2 nestles between the extracellular segment of VSDII and the pore extracellular loops in the third repeat (ECLIII), where it is stabilized by interactions with both protein regions and the branched N1372-glycan. ɩ-Conotoxin RXIA adopts an elongated conformation, spanning VSDI and VSDIV to wrap around the shoulder of the pore domain (PD). The bullet ant-derived toxin Pc1a (poneratoxin) exists as a transmembrane helix that inserts between VSDII and PDIII. Our findings, corroborated by functional characterizations, illustrate the remarkable diversity in peptide toxin binding modes and MOAs, providing a foundation for the rational design of selective Nav channel modulators.