Research Article

Structural basis of α-scorpion toxin action on Nav channels

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Science  22 Mar 2019:
Vol. 363, Issue 6433, eaav8573
DOI: 10.1126/science.aav8573

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How activation leads to gating

Voltage-gated sodium (Nav) channels are key players in electrical signaling. Central to their function is fast inactivation, and mutants that impede this cause conditions such as epilepsy and pain syndromes. The channels have four voltage-sensing domains (VSDs), with VSD4 playing an important role in fast inactivation. Clairfeuille et al. determined the structures of a chimera in which VSD4 of the cockroach channel NavPaS is replaced with VSD4 from human Nav1.7, both in the apo state and bound to a scorpion toxin that impedes fast activation (see the Perspective by Chowdhury and Chanda). The toxin traps VSD4 in a deactivated state. Comparison with the apo structure shows how interactions between VSD4 and the carboxyl-terminal region change as VSD4 activates and suggests how this would lead to fast inactivation.

Science, this issue p. eaav8573; see also p. 1278

Structured Abstract

INTRODUCTION

Members of the voltage-gated sodium (Nav) channel family are critical contributors to electrical signaling. Accordingly, they are targets of drugs, toxins, and mutations that lead to disorders such as epilepsy (Nav1.1 to Nav1.3 and Nav1.6), pain syndromes (Nav1.7 to Nav1.9), and muscle paralysis (Nav1.4 and Nav1.5). Nav 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 Nav 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 Nav1.7-NavPaS (human-cockroach) channel with and without AaH2 bound to illuminate the pharmacology of α-scorpion toxin action on Nav channels and gain insights into fast inactivation.

RATIONALE

For structural studies, we grafted the α-scorpion toxin receptor site from Nav1.7 onto the cockroach NavPaS channel chassis to ease challenges of producing human Nav channels. Specifically, we replaced VSD4 and a portion of the DI pore of NavPaS with related sequences from the human Nav1.7 channel. This protein engineering strategy permitted robust expression, purification, and complex formation between AaH2 and the Nav1.7-NavPaS chimeric channel. After cryo-EM structure determination of AaH2-bound and apo-Nav1.7-NavPaS channels to 3.5-Å resolution, we utilized traditional electrophysiological techniques to probe structure-function relationships in the related BgNav1 (cockroach), human Nav1.5 (cardiac subtype), and human Nav1.7 (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-Nav1.7-NavPaS channel structure uncovers a large S4 translation (~13 Å) 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 BgNav1, Nav1.5, and Nav1.7 channels recapitulate human disease-causing mutations and suggest that AaH2 has stabilized the fast-inactivation machinery of the Nav1.7-NavPaS 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 Nav 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 Nav channels. The pharmacology of α-scorpion toxins is further illuminated through an unexpected receptor site on VSD1 and pore-glycan interaction adjacent to VSD4.

Cryo-EM structures of a human-cockroach hybrid Nav channel in the presence and absence of the α-scorpion toxin AaH2.

(A) View of the AaH2-Nav1.7-NavPaS channel complex highlighting AaH2 (purple), VSD4 (green), gating charges (blue), the DIII-DIV linker (teal), CTD acidic residues (red), and the DI pore glycan (white). (B) Alternate view of the AaH2-channel complex [colored as in (A)] with the apo-Nav1.7-NavPaS channel structure (orange) superimposed. In the magnified view, the VSD4-based superposition highlights the extent of AaH2-induced translation of the S4 helix (AaH2 omitted for clarity).

Abstract

Fast inactivation of voltage-gated sodium (Nav) channels is essential for electrical signaling, but its mechanism remains poorly understood. Here we determined the structures of a eukaryotic Nav channel alone and in complex with a lethal α-scorpion toxin, AaH2, by electron microscopy, both at 3.5-angstrom resolution. AaH2 wedges into voltage-sensing domain IV (VSD4) to impede fast activation by trapping a deactivated state in which gating charge interactions bridge to the acidic intracellular carboxyl-terminal domain. In the absence of AaH2, the S4 helix of VSD4 undergoes a ~13-angstrom translation to unlatch the intracellular fast-inactivation gating machinery. Highlighting the polypharmacology of α-scorpion toxins, AaH2 also targets an unanticipated receptor site on VSD1 and a pore glycan adjacent to VSD4. Overall, this work provides key insights into fast inactivation, electromechanical coupling, and pathogenic mutations in Nav channels.

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