Diverse binding poses of agonistic neurotoxins on human Na_v1.6

Voltage-gated sodium (Na_v) channels are key targets of various venomous toxins. Deciphering the binding poses and mechanisms of action of representative toxins will help to dissect the functional mechanism of the channels and facilitate therapeutic development targeting Na_v channels^1,2. Here we present cryo-electron microscopy (cryo-EM) structures of distinct binding poses of three agonistic peptide toxins on the human Na_v1.6–β1 channel complex. The globular β-scorpion toxin Cn2 nestles between the extracellular segment of voltage-sensing domain (VSD) in the second repeat of the Na_v1.6 core α-unit (VSD_II) and the pore extracellular loops in the third repeat of the Na_v1.6 core α-unit (ECL_III), where it is stabilized by interactions with both protein regions and the branched N1372-glycan. Cone snail ι-conotoxin RXIA adopts an elongated conformation, spanning VSD_I and VSD_IV to wrap around the shoulder of the pore domain (PD). The bullet ant-derived toxin δ-paraponeritoxin-Pc1a exists as a transmembrane helix that stands between VSD_II and PD_III. Our findings, corroborated by functional characterizations, illustrate the diversity in peptide toxin binding poses and mechanisms of action, link stabilization of the up state of VSD_I or VSD_II to channel activation, and provide clues to the rational design of selective Na_v channel modulators.