Phrixotoxin-3 binds to three distinct antagonistic sites on human Nav1.6

• Published in: Cell research Vol. 35; no. 8; pp. 610 - 613
• Main Authors: Fan, Xiao, Chen, Jiaofeng, Huang, Xiaoshuang, Hou, Zhanfeng, Xie, Yuzhen, Li, Zigang, Yan, Nieng, Huang, Jian
• Format: Journal Article
• Language: English
• Published: Singapore Springer Nature Singapore 01.08.2025; Nature Publishing Group
• Subjects: 101/28; 631/337/470; 631/535/1258/1259; 82/83; 9/74; Antiepileptic agents; Binding sites; Biomedical and Life Sciences; Cell Biology; Channel gating; Channels; Chemical bonds; Data collection; Depolarization; Epilepsy; Excitability; Letter to the Editor; Life Sciences; Membrane potential; Membranes; Modulation; Molecular modelling; Muscles; Neurological diseases; Neurotoxins; Reagents; Sample preparation; Selectivity; Sodium; Sodium channels (voltage-gated); Tetrodotoxin; Toxins; Transient current
• ISSN: 1748-7838; 1001-0602; 1748-7838
• DOI: 10.1038/s41422-025-01141-4

Dear Editor, Voltage-gated sodium (Nav) channels govern the membrane excitability of neurons and muscles by mediating the rapid influx of Na+ ions in response to membrane depolarization.1 These functions are driven by the core Nav structure, which consists of a central pore domain (PD) and four surrounding voltage-sensing domains (VSDs).1 Among the nine human Nav subtypes (Nav1.1–1.9), Nav1.6, encoded by the SCN8A gene, is widely expressed in nervous systems, with particularly high density at the distal axon initial segment.2 Nav1.6 is critical in modulating the voltage threshold required for firing action potentials and is capable of generating resurgent currents, which are the small and transient currents elicited after depolarization at intermediate repolarizing potentials.3 These properties position Nav1.6 as a critical regulator of neuronal excitability, thus linking it closely to neurological disorders such as epilepsy.4 Despite its significance as a potential target for developing subtype-selective antiepileptic drugs,5 structural insights into Nav1.6 modulation by various reagents remain limited compared to other human Nav subtypes. Nav channels are subject to modulation by various natural toxins, which have historically been instrumental in elucidating channel structures and functions.1,6,7 For instance, the guanidinium neurotoxin tetrodotoxin has long been used to characterize and classify different Nav channels.7 In addition to directly interfering with ion permeation, some toxins alter the voltage dependence of the Nav for activation or inactivation, functioning as gating modifier toxins (GMTs).7 These GMTs typically bind to and trap VSDs in distinct functional states. PaurTx3 has been functionally characterized on some subtypes, such as Nav1.2 and Nav1.7, and functions as a potent Nav blocker by inducing a depolarizing shift in gating kinetics, a characteristic feature of typical GMTs.10 It has been reported that the toxin activity was influenced by protein lipidation, but the mechanism remains to be elucidated.11 In this study, we aim to investigate the molecular mechanism underlying PaurTx3 modulation and subtype selectivity across human Nav channels. The recombinant Nav1.6–β1 complex was purified following our reported protocol and incubated with PaurTx3 for cryo-sample preparation, data collection, and analysis.