Structural basis of α-scorpion toxin action on Nav channels

INTRODUCTION Members of the voltage-gated sodium (Na v ) channel family are critical contributors to electrical signaling. Accordingly, they are targets of drugs, toxins, and mutations that lead to disorders such as epilepsy (Na v 1.1 to Na v 1.3 and Na v 1.6), pain syndromes (Na v 1.7 to Na v 1.9), and muscle paralysis (Na v 1.4 and Na v 1.5). Na v channels contain four peripheral voltage-sensing domains (VSD1 to VSD4), which regulate the functional state of a central ion-conducting pore. Fast inactivation is an essential process that rapidly terminates Na + conductance, allowing excitable cells to repolarize and Na v channels to become available for reopening. Mutations that disrupt fast inactivation can cause devastating disease. Although the intracellular domain III-IV (DIII-DIV) linker and voltage-dependent conformational changes in VSD4 are known to be important for fast inactivation, structural details underlying the mechanism remain unclear owing to technical challenges. In this study, we used a potent α-scorpion neurotoxin, AaH2, that is known to target VSD4 to impede fast inactivation. We present cryo–electron microscopy (cryo-EM) structures of a hybrid Na v 1.7-Na v PaS (human-cockroach) channel with and without AaH2 bound to illuminate the pharmacology of α-scorpion toxin action on Na v channels and gain insights into fast inactivation. RATIONALE For structural studies, we grafted the α-scorpion toxin receptor site from Na v 1.7 onto the cockroach Na v PaS channel chassis to ease challenges of producing human Na v channels. Specifically, we replaced VSD4 and a portion of the DI pore of Na v PaS with related sequences from the human Na v 1.7 channel. This protein engineering strategy permitted robust expression, purification, and complex formation between AaH2 and the Na v 1.7-Na v PaS chimeric channel. After cryo-EM structure determination of AaH2-bound and apo-Na v 1.7-Na v PaS channels to 3.5-A resolution, we utilized traditional electrophysiological techniques to probe structure-function relationships in the related BgNa v 1 (cockroach), human Na v 1.5 (cardiac subtype), and human Na v 1.7 (peripheral nervous system) channels. RESULTS AaH2 wedges into the extracellular cleft of VSD4 to trap a deactivated state, analogous to a molecular stopper. Pharmacological trapping of VSD4 reveals state-dependent interactions of gating charges from the S4 helix and S4-S5 linker that bridge to acidic residues on the intracellular C-terminal domain (CTD). Our apo-Na v 1.7-Na v PaS channel structure uncovers a large S4 translation (~13 A) during VSD4 activation as a key molecular event leading to unlatching of the CTD and the fast-inactivation gating machinery. Analyses of structure-guided mutations in the BgNa v 1, Na v 1.5, and Na v 1.7 channels recapitulate human disease-causing mutations and suggest that AaH2 has stabilized the fast-inactivation machinery of the Na v 1.7-Na v PaS channel in a potential resting state. CONCLUSION Cryo-EM was used to visualize AaH2 in complex with the classic neurotoxin receptor site 3 on a hybrid eukaryotic Na v channel. Mechanistically, AaH2 traps VSD4 in a deactivated state, revealing an unanticipated interface through which DIV gating charges can couple to the CTD, DIII-DIV linker, and fast-inactivation gating machinery. We outline a structural framework that sheds light on the distinctive functional specialization of VSD4 and provides a deeper understanding of voltage sensing, electromechanical coupling, fast inactivation, and pathogenic mutations in human Na v channels. The pharmacology of α-scorpion toxins is further illuminated through an unexpected receptor site on VSD1 and pore-glycan interaction adjacent to VSD4.