v action on Na channels · vPaS channel chassis to ease challenges of producing human Na v...

RESEARCH ARTICLE SUMMARY◥

ION CHANNELS

Structural basis of a-scorpion toxinaction on Nav channelsThomas Clairfeuille, Alexander Cloake*, Daniel T. Infield*, José P. Llongueras*,Christopher P. Arthur*, Zhong Rong Li, Yuwen Jian, Marie-France Martin-Eauclaire,Pierre E. Bougis, Claudio Ciferri, Christopher A. Ahern†, Frank Bosmans†,David H. Hackos†, Alexis Rohou†, Jian Payandeh†

INTRODUCTION: Members of the voltage-gated sodium (Nav) channel family are criticalcontributors to electrical signaling. Accord-ingly, they are targets of drugs, toxins, andmutations that lead to disorders such as epi-lepsy (Nav1.1 to Nav1.3 and Nav1.6), pain syn-dromes (Nav1.7 to Nav1.9), andmuscle paralysis(Nav1.4 and Nav1.5). Nav channels contain fourperipheral voltage-sensing domains (VSD1 toVSD4), which regulate the functional state ofa central ion-conducting pore. Fast inactivationis an essential process that rapidly terminatesNa+ conductance, allowing excitable cells torepolarize and Nav channels to become avail-able for reopening.Mutations that disrupt fastinactivation can cause devastating disease. Al-though the intracellular domain III-IV (DIII-DIV)linker and voltage-dependent conformationalchanges in VSD4 are known to be importantfor fast inactivation, structural details underly-

ing the mechanism remain unclear owing totechnical challenges. In this study, we used apotent a-scorpion neurotoxin, AaH2, that isknown to target VSD4 to impede fast inac-tivation. We present cryo–electronmicroscopy(cryo-EM) structures of a hybrid Nav1.7-NavPaS(human-cockroach) channel with and withoutAaH2 bound to illuminate the pharmacologyof a-scorpion toxin action on Nav channels andgain insights into fast inactivation.

RATIONALE: For structural studies, we graftedthe a-scorpion toxin receptor site from Nav1.7onto the cockroach NavPaS channel chassis toease challenges of producing human Nav chan-nels. Specifically, we replaced VSD4 and a por-tion of the DI pore of NavPaS with relatedsequences from the human Nav1.7 channel.This protein engineering strategy permittedrobust expression, purification, and complex

formationbetweenAaH2and theNav1.7-NavPaSchimeric channel. After cryo-EM structure de-termination of AaH2-bound and apo-Nav1.7-NavPaS channels to 3.5-Å resolution, we utilizedtraditional electrophysiological techniques toprobe structure-function relationships in therelatedBgNav1 (cockroach), humanNav1.5 (car-diac subtype), and human Nav1.7 (peripheralnervous system) channels.

RESULTS:AaH2wedges into the extracellularcleft of VSD4 to trap a deactivated state, anal-ogous to amolecular stopper. Pharmacologicaltrapping of VSD4 reveals state-dependentinteractions of gating charges from the S4helix and S4-S5 linker that bridge to acidic res-

idues on the intracellularC-terminal domain (CTD).Our apo-Nav1.7-NavPaSchannel structure uncov-ers a large S4 translation(~13 Å) during VSD4 acti-vation as a key molecular

event leading to unlatching of the CTD and thefast-inactivation gating machinery. Analysesof structure-guided mutations in the BgNav1,Nav1.5, and Nav1.7 channels recapitulate humandisease-causing mutations and suggest thatAaH2 has stabilized the fast-inactivation ma-chinery of the Nav1.7-NavPaS channel in a po-tential resting state.

CONCLUSION: Cryo-EMwas used to visualizeAaH2 in complex with the classic neurotoxin re-ceptor site 3 on ahybrid eukaryoticNav channel.Mechanistically, AaH2 traps VSD4 in a deacti-vated state, revealing an unanticipated interface

throughwhichDIVgatingchargescan couple to the CTD, DIII-DIVlinker, and fast-inactivation gatingmachinery. We outline a struc-tural framework that sheds lighton the distinctive functional spe-cialization of VSD4 and providesa deeper understanding of volt-age sensing, electromechanicalcoupling, fast inactivation, andpathogenic mutations in humanNav channels. The pharmacol-ogy of a-scorpion toxins is furtherilluminated through an unex-pected receptor site on VSD1 andpore-glycan interaction adjacentto VSD4.▪

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Clairfeuille et al., Science 363, 1302 (2019) 22 March 2019 1 of 1

The list of author affiliations is available inthe full article online.*These authors contributed equally to this work.†Corresponding author. Email:[email protected] (C.A.A.);[email protected] (F.B.); [email protected] (D.H.H.); [email protected] (A.R.); [email protected] (J.P.)Cite this article as T. Clairfeuille et al.,Science 363, eaav8573 (2019).DOI: 10.1126/science.aav8573

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Cryo-EM structures of a human-cockroach hybrid Nav channel in the presence and absenceof the a-scorpion toxin AaH2. (A) View of the AaH2-Nav1.7-NavPaS channel complex highlightingAaH2 (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 [coloredas in (A)] with the apo-Nav1.7-NavPaS channel structure (orange) superimposed. In the magnifiedview, the VSD4-based superposition highlights the extent of AaH2-induced translation of the S4helix (AaH2 omitted for clarity).

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RESEARCH ARTICLE◥

ION CHANNELS

Structural basis of a-scorpion toxinaction on Nav channelsThomas Clairfeuille1, Alexander Cloake1,2*, Daniel T. Infield3*, José P. Llongueras4*,Christopher P. Arthur1*, Zhong Rong Li5, Yuwen Jian6,Marie-France Martin-Eauclaire7, Pierre E. Bougis7, Claudio Ciferri1,Christopher A. Ahern3†, Frank Bosmans8†, David H. Hackos6†,Alexis Rohou1†, Jian Payandeh1†

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 aeukaryotic Nav channel alone and in complex with a lethal a-scorpion toxin, AaH2, by electronmicroscopy, 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 chargeinteractions 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 a-scorpion toxins, AaH2also targets an unanticipated receptor site onVSD1 and a pore glycan adjacent to VSD4.Overall,this work provides key insights into fast inactivation, electromechanical coupling, andpathogenic mutations in Nav channels.

Voltage-gated sodium (Nav) channels initiateand propagate action potentials in excitablecells (1–3). After voltage-dependent open-ing,Nav channels undergo fast inactivationon the millisecond time scale to terminate

Na+ conductance (3, 4). Fast inactivation is anessential hallmark of Nav channel function thatallows cells to repolarize and Nav channels tobecome readily available for reactivation (1, 3, 5).Venomous animals have evolved an arsenal oftoxins that disrupt this process to immobilize preyor predators, highlighting the importance of fastinactivation across the animal kingdom (6, 7). Inhumans, mutations that even subtly affect fastinactivation can cause epilepsy, cardiac arrhyth-mias, muscle disorders, or pain syndromes (2, 3).In 1952, Hodgkin and Huxley postulated that

Nav channels contain three “m” gating particlesresponsible for channel opening and one slower“h” gating particle associated with inactivation(8). Nav channels are now known to contain fourhomologous repeat domains (DI toDIV) inwhich

four voltage-sensing domains (VSDs) surround acentral pore module (PM) in a domain-swappedarrangement (9–12). Voltage-sensing domain IV(VSD4) has been established to play a specializedrole in initiating fast inactivation (13–17), andtoxins that target the extracellular surface ofVSD4 modify this process (18–20). Mutagenesisstudies identified an isoleucine-phenylalanine-methionine (IFM) motif within the intracellularDIII-DIV linker as an important part of the fast-inactivation gating machinery, leading to a con-ceptual hinged-lid model of fast inactivation(21, 22). Fast inactivation remains enigmatic,however, because the structural mechanismof coupling between VSD4 and the DIII-DIVlinker has not been described; therefore, howtoxins or disease mutations might modify thisprocess also remains unknown.Nav channels share a conserved architecture

with other voltage-gated ion channels (23, 24).The VSDs are four-helix bundles (S1 to S4) thatsensemembranepotential usingpositively chargedresidues found in a repeating arginine-X-X (RXX)motif along the S4 helix (9, 25, 26). The S5 and S6helices from each homologous domain togetherform the central PM (10, 24). Upon membranedepolarization, VSDs activate through the out-ward movement of S4-gating charges across anarrow hydrophobic constriction (25, 27, 28),which couples through intracellular S4-S5 linkersto dilate the S6 PM bundle crossing and initiateNa+ conductance (29–31). Studies tracking S4movements indicate that rapid VSD1 to VSD3activation is requisite to open the central PMgate,where VSD4 activation is slower but necessaryand sufficient to initiate fast inactivation (15, 17).Sequence asymmetry has permitted the functional

specialization of Nav channel VSDs, but the basisfor their asynchronous activation remains un-clear (15–17, 32, 33). Indeed, direct observationof the structural transitions correlated with volt-age sensing, electromechanical coupling, and fastinactivation remains technically challenging owingto the negative membrane potentials required tomaintain VSDs in a deactivated state.Peptide toxins that target VSDs have been

useful tools for elucidating the complex gatingproperties of Nav channels because they can alterthe stability of open, closed, or inactivated states(34–36). In particular, a-scorpion toxins impedeNav channel fast inactivation to sustain sodiuminflux, causing prolonged action potentials anddecreased firing frequency in vivo, which lead toparalysis, cardiac arrhythmia, or death (6, 7, 37, 38).Onlyminute quantities of toxin II (AaH2) from theAndroctonusaustralisHector “mankiller” scorpionare required to impart a lethal dose (39, 40). AaH2is a 64–amino acid peptide stapledby four disulfidebonds into a compact b1-a1-b2-b3 scaffold thatcan modify multiple mammalian Nav channelsubtypes with high potency (40, 41). Historicallycalled neurotoxin receptor site 3 (42), the bindingsite for a-scorpion toxins has beenmapped acrossthe extracellular surface of VSD4 and onto theadjacent DI PM (43–45). Functionally, a-scorpiontoxins delay ~30% of total gating charge move-ment in Nav channels, presumably by trappingVSD4 in a deactivated state (14, 18). Thus, clarify-ing the details of AaH2 action on Nav channelsshould provide insights into fast inactivation androutes to design new channel modulators.Structure determination of eukaryotic Nav

channels has become possible with breakthroughsin cryo–electron microscopy (cryo-EM) (46). Thecockroach NavPaS and eel Nav1.4 channels pro-vided the first high-resolution structural templates(10, 11); however, NavPaS remains recalcitrant tofunctional recordings, and eelNav1.4was obtainedfrom native sources. A recent human Nav1.4channel structure has confirmed the utility ofNavPaS and eel Nav1.4 as surrogate structuralmodels but also highlighted the challenges ofproducing neuronal Nav channel subtypes, likeNav1.7 (12, 47). Human genetic studies have iden-tified loss-of-function mutations in Nav1.7 thatresult in congenital insensitivity to pain (48, 49).These findings have motivated efforts to developNav1.7-selective inhibitors that could overcomethe liabilities of opioid analgesics (50). How-ever, clinically available Nav channel inhibitorslack molecular selectivity, and the discovery ofsubtype-selective antagonists remains challenging(51). Focusing on Nav1.7 as a model system, wehumanized VSD4 of NavPaS to capture an AaH2toxin–channel complex at high resolution. Ourdata reveal the structural basis of a-scorpiontoxin action on Nav channels, which provides in-sight into the mechanisms of voltage sensing,electromechanical coupling, and fast inactiva-tion operating in Nav channels.

AaH2 action on Nav channels

To assess the activity of AaH2 purified from the ve-nom of the Androctonus australis Hector scorpion,

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Clairfeuille et al., Science 363, eaav8573 (2019) 22 March 2019 1 of 14

1Department of Structural Biology, Genentech Inc., SouthSan Francisco, CA, USA. 2Department of Physics, Universityof Oxford, Oxford OX1 3PU, UK. 3Department of MolecularPhysiology and Biophysics, The University of Iowa, Iowa City,IA, USA. 4Department of Physiology, Johns HopkinsUniversity School of Medicine, Baltimore, MD, USA.5Department of Biomolecular Resources, Genentech Inc.,South San Francisco, CA, USA. 6Department ofNeuroscience, Genentech Inc., South San Francisco, CA,USA. 7Aix Marseille Université, CNRS, LNC, UMR 7291, 13003Marseille, France. 8Department of Basic and Applied MedicalSciences, Ghent University, 9000 Ghent, Belgium.*These authors contributed equally to this work.†Corresponding author. Email: [email protected](C.A.A.); [email protected] (F.B.); [email protected] (D.H.H.); [email protected] (A.R.); [email protected] (J.P.)

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we first measured its effect (median effective con-centration, EC50) on human Nav1.2 and Nav1.7channels at a membrane potential that stabil-izes the closed state (−100 mV). AaH2 impairedfast inactivation at doses expected for a-scorpiontoxins, for which the potency of modulation high-lights the potential of AaH2 to target neuro-toxin receptor site 3 across multiple Nav channelsubtypes (Fig. 1, A and B; EC50 of 0.72 ± 0.59and 51.7 ± 1.5 nM for Nav1.2 and Nav1.7, respec-tively). AaH2 started to inhibit peak currents ofNav1.2 and Nav1.7 at higher toxin concentra-

tions (Fig. 1, A and B). AaH2 action on Nav1.7was state-dependent, as expected, with the toxinbeing ~100-fold less potent at holding potentialsthat stabilize VSD4 in an activated conformation(Fig. 1C).

Generation of an AaH2-Nav1.7-NavPaSchannel complex

Despite our interest in Nav1.7-selective modula-tors (50–55), studies of AaH2 in complex withVSD4 were initially unsuccessful because puri-fication of full-length human Nav1.7 remains

challenging (12, 47). We considered a proteinengineering approach because VSDs that aretransferred onto related channels can retaintheir pharmacological and structural properties(23, 56, 57). Indeed, we previously used thisapproach to visualize VSD4 in complex withthe Nav1.7-selective antagonist GX-936 in thecontext of a human-bacterial Nav channel chimera(52). We reasoned that the cockroach NavPaSchannel (10) might facilitate the production of aNav1.7-NavPaS hybrid channel suitable to allowvisualization of an AaH2 complex.


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Fig. 1. Characterization and cryo-EM structure of the AaH2-VSD4-NavPaS channel complex. (A) Sodium current traces from human Nav1.7measured during a 0-mV pulse from a holding potential of −100 mV (black)with different doses of AaH2 (purple and green). (B) EC50 measurementsof AaH2 from human Nav1.2 (gray) and human Nav1.7 (black) channelsmeasured at a holding potential of −100 mV. (C) EC50 measurements ofAaH2 from human Nav1.7 channels measured at a holding potential of−120 mV (black) or −40 mV (gray). (D) Schematic of the Nav1.7 VSD4–DI-S5–NavPaS channel. The DIII-DIV linker is shown in cyan, and portions

humanized to the Nav1.7 sequence are shown in green. N-terminal domain(NTD) and CTD are indicted. (E) Differential scanning fluorimetry of purifiedWT-NavPaS and VSD4-NavPaS channels (shown here are data for the VSD4–DI-S5–NavPaS construct) in the absence or presence of GX-936 or AaH2.dl330/350, first derivative of the fluorescence ratio change. (F) Side view(sectioned) of the single-particle cryo-EM reconstruction of the AaH2–VSD4–DI-S5–NavPaS channel complex (hereafter called the VSD4-NavPaSchannel). (G) Top view of the single-particle cryo-EM reconstruction of theAaH2-VSD4-NavPaS channel complex.

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Replacing VSD4 of NavPaS with VSD4 fromhumanNav1.7 enabled high-yield production ofa chimeric channel (Fig. 1D and fig. S1, A to C).Because Na+ currents could not be recordedfrom this construct, we used differential scan-ning fluorometry and the potent small-moleculeinhibitor GX-936 to confirm reconstitution of arelevant Nav1.7 receptor site (Fig. 1E and fig. S1D)

(52, 58). However, in contrast to GX-936, AaH2did not substantially thermostabilize this VSD4-NavPaS chimeric channel (fig. S1D). On the basisof neurotoxin receptor site 3 mapping studiesand the domain-swapped architecture of Navchannels (10–12, 43–45), we grafted the DI-S5helix from human Nav1.7 onto a second chimericconstruct (Fig. 1D). AaH2 appreciably thermo-

stabilized this optimized VSD4–DI-S5–NavPaSchannel construct and coeluted with the purifiedprotein as a complex (Fig. 1E and fig. S1D). Wepurified this VSD4–DI-S5–NavPaS chimeric chan-nel (hereafter called VSD4-NavPaS) in the de-tergent digitonin supplemented with cholesterylhemisuccinate and AaH2 and obtained a single-particle cryo-EM reconstruction of the toxin-channel complex with a nominal resolution of~3.5 Å at the AaH2-VSD4 receptor site (figs. S2,A and B, and S3 and table S1). To enable a directcomparison, we also analyzed single-particle cryo-EM reconstruction of a similarly purified apo-VSD4-NavPaS chimeric channel, in the absenceof AaH2 toxin, at a nominal resolution of ~3.4 Åat the VSD4 receptor site (fig. S2, C and D, andtable S1).

Overall structure of the AaH2-VSD4-NavPaS channel complex

The AaH2-VSD4-NavPaS channel complex re-sembles a four-point star, in which two tips ofthe star are occupied by toxin (Fig. 1, F and G,and Movie 1). AaH2 is wedged into an extra-cellular cleft formed by VSD4 and the DI PM(Fig. 1F), where it binds to VSD4 through con-tacts across the S1-S2 and S3-S4 loops (Fig. 2, Aand B). AaH2 utilizes C-terminal residues Arg62

andHis64 to engage the DI PM, while N-terminalresidues contact a glycan moiety that decoratesthe DI-pore turret loop structure (Fig. 3, A andB). The overall architecture of the AaH2-VSD4-NavPaS complex agrees with expectations fromneurotoxin receptor site 3 mapping studies(43–45), although participation of a PM glycanwas not previously appreciated.Unexpectedly, a second AaH2 toxin is seen

bound to VSD1 (Fig. 1G). Density for this AaH2molecule is less defined than that for the toxinthat engages VSD4 (Fig. 1G and fig. S2B), sug-gesting a lower-affinity AaH2-VSD1 interaction.Because a-scorpion toxin binding to VSD1 hasnot been previously reported, we carefully refinedan AaH2 model into this density (fig. S3) andclosely examined our apo-VSD4-NavPaS chimericchannel structure (figs. S1, A and B, and S2, Cand D). The availability of toxin-bound and apo-VSD4-NavPaS channel structures provides a rareopportunity to evaluate the consequences of AaH2binding, including the direct observation of VSD4activation.

Activation and gating-chargetransfer in VSD4

The structural basis of voltage sensing in VSD4has remained elusive but can nowbe appreciatedby comparingAaH2-bound and apo-VSD4-NavPaSchannel structures (Fig. 2, A to E, and Movie 2).In the apo-VSD4-NavPaS channel, VSD4 is foundin an activated conformation (Fig. 2A). Typical ofmost VSDs visualized at 0-mV potential (fig. S4,A to C) (10–12, 23, 24, 52, 59), the R5 (i.e., fifth)gating charge is found just beneath the conservedS2 phenylalanine (Phe1547) within the intracellularvestibule (Fig. 2A and fig. S4B). Physiologicalmeasurements suggest that when R5 is housedin this location and the R1 to R4 gating charges


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Fig. 2. Activation and gating-charge transfer in VSD4. (A) Side view of VSD4 from the apo-VSD4-NavPaS channel showing the ENC (red), INC (red), HCS (orange), and gating charges (R1 to R5 andK6, blue). Shown on the right are the positions of S4 gating charges relative to the AaH2-VSD4-NavPaS channel complex, with approximate distances between “equivalent C⍺ atoms” indicated forreference. (B) Side view of VSD4 from the AaH2-VSD4-NavPaS channel showing AaH2 (pink) andgating charges (R1 to R5 and K6, blue). R5 gating charge interactions with the intracellular CTD(gray) are shown. (C) Top view of the superposition of apo-VSD4 (gray) and AaH2-VSD4 (green). TheR1 to R5 and K6 gating charges are rendered as spheres (but only to Cb for clarity); R3 in bothmodels has been colored blue for reference. Phe1547 from the HCS is shown as sticks; S3-S4 loopsand AaH2 have been omitted for clarity. (D) Side view with S3-S4 loops shown, otherwise similar to(C). (E) Close-up view from the area outlined by dashed lines in (D). Phe1583 (S3) and Glu1589 sidechains are shown for reference, and AaH2 is omitted for clarity. Single-letter abbreviations for theamino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys;L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.


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reside above the hydrophobic constriction site(HCS), VSD4 is in an activated conformation(25, 52, 59). Superposition of the available GX-936–VSD4–NavAb structure corroborates thisassignment because GX-936 is a potent antag-onist known to stabilize Nav1.7 VSD4 in anactivated state (fig. S4C) (52). By contrast, theAaH2-VSD4-NavPaS channel structure revealsVSD4 in a deactivated state because only the R1and R2 gating charges are located above theHCS within the extracellular vestibule (Fig. 2, Bto D, and fig. S4D). Successful pharmacologicaltrapping of the deactivated conformation ofVSD4 under solution conditions likely reflectsthe high potency of AaH2 for Nav1.7 and ourchimeric channel construct (Fig. 1, C and E).Cataloging the S4 gating charge locations pro-

vides insight into voltage sensing because theS4 helix translates ~13 Å (equivalent Ca-backboneatoms) between the apo-VSD4–activated andAaH2-VSD4–deactivated states (Fig. 2, A to D,andMovie 2). Each S4 arginine side chain relocatestwo positions along the gating charge–transferpathway, where R3 and R4 toggle between theextracellular negative-charge cluster (ENC) andthe intracellular negative-charge cluster (INC)(Fig. 2, A to D). Porting of these two gatingcharges across the HCS is consistent with phys-iological measurements where a-scorpion toxinsimmobilize ~30% of total Nav channel gatingcurrents (14, 18), equivalent to the movement ofabout two elementary charges across the mem-brane electric field. In the apo-VSD4–activatedstate (Fig. 2A), R1 extends toward the DI-S5 helixdipole; R2 and R3 engage Gln265 (DI-S5) andapproach Glu1524 on the S1 helix; R4 interactswith Asn1517 (S1) and potentially Asn1540 (S2) abovethe HCS; R5 pairs with Asp1571 (S3) below theHCS; and gating charge K6 aligns along Trp1567

(S3), a side chain conserved inNav channel VSDs.In comparison, in the AaH2-VSD4–deactivatedstate (Fig. 2B), R1 pairswithAsn1540 (S2); R2 bondswith Asn1517 (S1) above the HCS; R3 is coordinatedto Glu1550 (S2) and Asp1571 (S3) below the HCS; R4aligns between Asp1507 (S1) and Trp1567 (S3); R5extends beyond the vestibule of VSD4 towardGlu1431-NavPaS on the intracellular C-terminaldomain (CTD); andK6 similarly reaches to Gln1461-NavPaS on the CTD. Overall, the apo-VSD4–activated and AaH2-VSD4–deactivated statestructures appear to represent physically rea-sonable models of activation and gating-chargetransfer inVSD4 (Fig. 2, A to E, and fig. S4, A toD).Superposition of apo-VSD4–activated and

AaH2-VSD4–deactivated state structures illus-trates the mechanics of VSD4 activation in Navchannels (Fig. 2, C to E, and Movie 2). In theAaH2-VSD4–deactivated state, the S4 helix existsmainly in a 310-helical conformation, which servesto align gating charges for activation through thecentral gating pore (Fig. 2, B and C). Notably, wefind the R5 gating charge positioned to interactwith an acidic surface patch on the intracellularCTD (Fig. 2B). In the apo-VSD4–activated state,the S4 remains in a 310-helical conformation acrossthe gating pore but adopts an a-helical conforma-tion above the HCS (Fig. 2, A, C, and D). When di-

rectly superimposed, small deviations over the S1to S3 core region indicate that the membrane elec-tric field works to translate S4 gating charges ac-ross the HCS with minimal global rearrangementsof VSD4 during activation (Fig. 2, C to E). Phe1583

emerges as a hinge point on the S3helix that allowsthe S3-S4 loop to undergo substantial displacementduring VSD4 activation gating (Fig. 2, D and E, andfig. S4, A, C, andD). This S3-S4 loop displacementhas clear implications for state-dependent bindingof AaH2 to neurotoxin receptor site 3.

Determinants of AaH2 binding toneurotoxin receptor site 3

The structure of theAaH2-VSD4-NavPaS channelcomplex illuminates the molecular details ofa-scorpion toxin binding, polypharmacology,and antagonism of Nav channels. Akin to a mo-lecular doorstop, the triangular-shaped AaH2toxin makes multipoint contacts across VSD4and the neighboring DI PM (Fig. 3, A to D). Thecompact b1-a1-b2-b3 fold of AaH2 is required towedge into the receptor site, rationalizing whyits four disulfide bonds are essential for potency(60). High surface and electrostatic complemen-tarities appear to dominate contributions tocomplex formation (Fig. 4, A to C), but AaH2also targets potential subtype-selective Navchannel determinants (Fig. 3, B to D and G,and figs. S5 and S6). Consistent with a high-affinity complex, a considerable surface areaof AaH2 (~712 Å2) is buried by the neurotoxinreceptor site 3 interaction, where the channelinterface can be divided into four regions: the DIPM, a PM glycan, the S1-S2 loop, and the S3-S4loop (Fig. 3, A to D, and Movie 1).AaH2 uses both its rigid core scaffold and loop

elaborations to target neurotoxin receptor site 3.Structure-activity relationship studies have sug-gested that Arg62 and His64 on the C-terminalsegment (CTS) of AaH2 are critical for potentmodulation of Nav channels (40, 61, 62). In theAaH2-VSD4–deactivated state, Arg62 hydrogenbonds directly to the carbonyl of Gln265 (DI-S5helix) and the side chain of Glu1589 (S3-S4 loop),while His64 forms a close interaction networkwith a constellation of DI PM side chains in-cluding Asn270, His273, and Gln345-NavPaS (Fig. 3,A and B). Because Gln265, Asn270, and His273 areresidues grafted from human Nav1.7 onto theNavPaS channel chassis required for AaH2-mediated stabilization (fig. S1, A andD), these DIPM interactions likely lock AaH2 into a produc-tive receptor site complex and together mayrepresent determinants of toxin potency andselectivity (fig. S6). Comparison to availablestructures of AaH2 bound or unbound to amono-clonal antibody fragment further emphasizes akey role for Arg62 and His64 in targeting neuro-toxin receptor site 3 (Fig. 3, E and F) (41, 63) andsuggests that sequestering of Arg62 and His64 issufficient for antibody-mediated neutralization ofthis lethal toxin (Fig. 3F).The N-terminal reverse turn (RT) and CTS of

a-scorpion toxins are known to be important forneurotoxin receptor site 3 binding (61, 64, 65). InAaH2, this region is wedged alongside the elab-

orated structure of the DI PM (Fig. 3, A and B).Asp9 (RT), Val10 (RT), and Arg56 (CTS) contact anextended PM glycan that shields a hydrophobicsurface on the turret loop structure (Fig. 3, A andB, and figs. S5 and S6). This glycosylation em-anates from the NavPaS channel chassis (Asn

330-NavPaS) and is conserved inmost mammalian Navchannel subtypes (fig. S6). Curiously, humanNav1.7lacks this glycosylation locus (fig. S6), establish-ing that toxin contacts to the PM glycan are notstrictly required for potent AaH2-mediated Navchannel modulation (Fig. 1B).The S1-S2 loop region of VSD4 is contacted by

the CTS and b2-b3 loop of AaH2 (Fig. 3, A and C).Tyr42 (b2-b3 loop) bonds to Gln1530 (S1-S2 loop),as Gly59-Pro60 (CTS) frames theGln1196 side chain(Fig. 3C). The carbonyl of Ala39 (b2-b3 loop) en-gages Tyr1537 (S2), as Ser40-Pro41 covers the Tyr1203

side chain (Fig. 3C). Targeting of Tyr1537 by AaH2is notable because this residue is a hotspot forNav1.7-selective antagonists (GX-936) and Nav1.1-selective activators (Hm1a) (52, 66). However,limited side-chain engagement along the S1-S2region also rationalizes the ability of AaH2 tomodulate multiple Nav channel subtypes.The S3-S4 loop region of VSD4 forms a state-

dependent interface that is engaged by the RT,b2-b3 loop, and CTS of AaH2 (Fig. 3, A and D).Asn44 (b2-b3 loop) bonds directly to the carbonylof Phe1583 (S3), as Phe15 and Trp38 (b2-b3 loop)


Movie 1. AaH2 receptor sites on the Nav1.7VSD4-NavPaS channel. Views of the molecularinteractions between AaH2 and the Nav1.7VSD4-NavPaS chimeric channel.

Movie 2. VSD4 activation in the Nav1.7VSD4-NavPaS channel. Trajectory (morph)between the AaH2-bound and AaH2-free (apo)structures of the VSD4-NavPaS channel. AaH2is not shown for clarity.


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RT

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NH2NH2+

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NH2+

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NH2O

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Fig. 3. AaH2 in complex with neurotoxin receptor site 3. (A) Side view ofthe AaH2-VSD4 receptor site complex. Structural elements of AaH2 areindicated, and Arg62 and His64 side chains are shown as sticks for reference.(B) AaH2 interface with the DI PM and PM glycan. Dashed lines indicate likelydirect-bonding interactions. (C) AaH2 interactions with the S1-S2 loopregion. (D) AaH2 interactions with the S3-S4 loop region. (E) Superpositionof an unbound AaH2 crystal structure [orange; Protein Data Bank (PDB)1PTX] with the AaH2-VSD4-NavPaS channel complex (purple) reveals differ-ences in the CTS region upon Nav channel binding; here, the channel is omitted

for clarity. (F) Structure of AaH2 bound to a neutralizing antibody 4C1 fragment(PDB 4AEI). Heavy and light chains are shown in blue and yellow surfacecoloring, respectively. (G) EC50 measurements of AaH2 from human Nav1.7channels. Shown are WT Nav1.7 (gray), mutants in the DI PM (light orange), theS1-S2 loop region (blue), and the S3-S4 loop region (green), colored accordingto the structure model represented in (A). Error bars represent mean ± SEM.(H) EC50 measurements of syntheticWTand mutant AaH2 toxins tested on theWT Nav1.7 channel. Schematics of non-alanine side-chain chemistries areshown for reference. Error bars represent mean ± SEM.


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make flanking hydrophobic and van der Waalsinteractions (Fig. 3D). Notably, Phe1583 demarksthe extracellular end of the S3 helix in the de-activated state of VSD4 (Figs. 2D and 3D), ademarcation not seen in all VSD4-activated statestructures (fig. S4, A and C) (11, 12, 52). Theseobservations establish that Asn44 targets a keystate-dependent interaction on VSD4. Similarly,Thr13 (RT) of AaH2 coordinates Asp1586 (S3-S4loop), as Arg62 (CTS) and the Cys63 carbonyl (CTS)form a networkwithGlu1589 (S3-S4 loop), whereasAsp1586 and Glu1589 can form part of the S3 helixin available VSD4-activated state structures (fig.S4, A and C) (11, 12, 52). Overall, the structuraldeterminants of AaH2 binding revealed herealign with prior receptor site–mapping studiesbut provide a deeper understanding of the statedependence of toxin modulation.

AaH2 traps VSD4 in a deactivated state

The AaH2-VSD4–deactivated and apo-VSD4–activated state structures clarify themechanismbywhich a-scorpion toxins impede VSD4 activation.

In the deactivated state, AaH2 wedges into theextracellular cleft of VSD4 to make multipoint-bridging contacts across the VSD4 loops and DIPM (Figs. 3A and 4, A and B, and Movie 1). Thisinvokes a simple physical mechanism to antago-nize VSD4 activation: AaH2 acts as a molecularstopper to prevent outward movement of the S4voltage sensor.In the VSD4-deactivated state, the receptor

site presents an intense electronegative surfacethat is matched by an electropositive surface onAaH2, indicating a role for electrostatic steeringin complex formation (Fig. 4, A and B). The largefootprint that AaH2 occupies across neurotoxinreceptor site 3 (~712 Å2) must also drive toxinpotency for the deactivated state. By contrast, theVSD4-activated state undergoes substantial con-formational and electrostatic remolding upon S4activation (Figs. 2, C to E, and 4C), rationalizingwhy AaH2modulation is state-dependent (Fig. 1C).The S4 helix undergoes considerable displace-

ment upon VSD4 activation (Figs. 2, C to E, and4, D to F). On superposition, a severe clash occurs

between the activated S4 helix and AaH2, ex-plainingwhy the S4 is translateddownward (~13Å)in the toxin-bound complex (Fig. 4, E and F).Indeed, Arg62 of AaH2 occupies a similar DI-S5helix coordination site as the R1 gating chargein the apo-VSD4–activated state, demonstratingthat the toxin physically occupies a locale visitedby gating charges during VSD4 activation (Fig. 4,D and E). These observations rationalize whyAaH2 action is state-dependent and raise specula-tion that the fat-tailed scorpion has evolved Arg62

in AaH2 as a gating charge mimetic.The S3-S4 loop is amajor binding determinant

for a-scorpion toxins (Fig. 3, D and G); however,this loop undergoesmarked rearrangements uponVSD4 activation (Fig. 4F and fig. S4, A, C, and D).Accordingly, AaH2 uses Thr13, Asn44, Arg62, andCys63 to focusmultipoint coordination across theS3-S4 loop while simultaneously targeting inter-actions that are only available in the deactivatedstate (Phe1583, Asp1586, and Glu1589) (Fig. 3D).AaH2 binding therefore sterically prevents theS4 helix and S3-S4 loop from undergoing the


A C apo-VSD4-activatedVSD4 surface

D

11Å

R1

R62

R2

R1

R3

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S4 helix “down”

apo -VSD4, AaH2 superimposed

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D1586E1589

DI-S5

E1534

E1524

E1525

R62

E

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AaH2-VSD4-deactivatedAaH2 surface

B

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AaH2-VSD4-deactivatedVSD4 surface

S1-S2 loop

S3 S4

S3-S4 loop

AaH2

S1-S2 loop

S3-S4 loop S3-S4 loop

S1-S2 loop

Fig. 4. VSD4 voltage-sensor trapping by AaH2. (A) Extracellular view ofthe AaH2-VSD4 complex, where VSD4 and the DI PM are shown inelectrostatic surface representation. Arg62 from AaH2 is shown in purplestick representation for reference. (B) Same view as in (A), with anelectrostatic surface of AaH2 rendered and VSD4 shown in cartoon. Selectresidues on VSD4 are shown in stick representation for reference. Themagenta outline indicates the surface border of AaH2. (C) The apo-VSD4structure is shown in electrostatic surface representation, in a similar view

as in (A). (D) Side view of the AaH2-VSD4 complex. Only Arg62 of AaH2and the R1 gating charge are shown in stick representations for clarity.(E) Side view of the apo-VSD4 structure with the AaH2-VSD4 complexsuperimposed (although VSD4-deactivated is removed for clarity).A clash between Arg62 (AaH2) and R1 (S4) side chains is indicated by dots.(F) Similar to (E), but a different view, and with AaH2 in purple surfacerepresentation. Arrows point to clashes between AaH2 and the VSD4-activated state (gray) structure.


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appreciable conformational changes and electro-static remolding required for VSD4 to achievean activated state (Fig. 4, D to F). These struc-tural observations collectively explain the state-dependence of AaH2 and impose a voltage-sensortrapping mechanism.

Electrostatics affect toxin binding andVSD4 trapping

Guided by the AaH2-VSD4-NavPaS complexstructure, we set out to probe details of AaH2interaction with neurotoxin receptor site 3 inthe human Nav1.7 channel. Alanine mutationswere generated for the equivalent three DI PMand four VSD4 side chains found to contactAaH2 directly (Fig. 3, A to D and G). Only theAsp1586→Ala (Asp1586Ala) mutation appreciablyshifted the EC50 (Fig. 3G and fig. S7, A and B),

confirming that this acidic residue on the S3-S4loop is a binding hotspot (45). However, theAsp1586Arg mutant Nav1.7 channel also returneda considerable EC50 for AaH2 (Fig. 3G and fig.S7B), consistent with the large surface area AaH2uses for binding and suggesting that a-scorpiontoxins have evolved to engageNav channels robustly.We next sought to interrogate the Nav1.7

channel receptor site through the study of AaH2variants. The His64Ala-AaH2 toxin displayed a~fourfold loss in potency (Fig. 3H and fig. S7C),in line with the PM single-point mutations thathad only a minor impact (Fig. 3G). By contrast,the Arg62Ala-AaH2 mutant significantly reducedtoxin potency, ~80-fold (Fig. 3H and fig. S7C).As citrulline (Cit) is a neutral amino acid that isnearly isosteric with arginine (67), we examinedan Arg62Cit-AaH2 toxin analog and measured an

intermediate ~40-fold decrease in potency (Fig. 3Hand fig. S7C). Further substitutions at position62 that increased (hArg, homoarginine) or de-creased (nArg, norarginine; and Agp, 2-amino-3-guanidinopropionic acid) the span of theguanidino group by one or two methylene unitsrelative to arginine returned nearly identicalpotencies to the wild-type (WT) toxin (Fig. 3H),suggesting that Arg62 in native AaH2 is wellpositioned to antagonize Nav channels. Overall,Arg62 is an important contributor to AaH2 potencyand appears to impart an electrostatic com-ponent to help impede S4 activation and trapVSD4 in the deactivated state (Fig. 3H).

AaH2 can bind to VSD1

a-Scorpion toxin action on Nav channels hasbeen the subject of investigation for more than


H64

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DII-

pore

A C

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Fig. 5. AaH2 bound to a VSD1 receptor site. (A) Side view of the AaH2-VSD1 complex. Arg62 and His64 from AaH2 are shown as sticks forreference. (B) Side view of the activated VSD1, with AaH2 removed forclarity. ENC (red), INC (red), HCS (orange), and gating charges (R1 to R4,purple) are indicated. (C) AaH2-VSD1 (orange-blue) and AaH2-VSD4(purple-green) complexes are superimposed based only on the VSD

scaffold. (D) Close-up view of the VSD1 receptor site highlighting AaH2interactions with Tyr166 (S2) and Asp219 (S3-S4 loop). (E) AaH2 wasapplied at 500 nM to WT, VSD1-Asp220Arg, and VSD4-Asp1670Arg BgNav1channels. Representative current traces from Xenopus oocytes at 0.2 Hz(upper) and 1.0 Hz (lower) shown at 35 s (orange) and 150 s (blue) aftertoxin application.


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50 years (37, 38), so it was initially surprising tofind a second AaH2 toxin molecule bound to theVSD4-NavPaS channel (Figs. 1G and 5, A to D,and Movie 1). The weaker density for the AaH2-VSD1 complex suggests a low-affinity interaction(Fig. 1G and fig. S2B), which may explain why ithas previously gone undetected, and calls intoquestion the physiological relevance of this prim-itive VSD1 receptor site. Still, understanding thedeterminants of AaH2 binding to VSD1 will il-luminate the structural basis of toxin polyphar-macology and may reveal principles to designnew Nav channel modulators.AaH2 binds to an activated conformation of

VSD1 under our experimental conditions (Fig. 5,A and B). In the AaH2-VSD1 complex, R4 formssalt bridges with acidic residues below the HCS,permitting the R1 to R3 gating charges to projectinto the vestibule to engage typical ENC inter-actions (Fig. 5B). Global changes in VSD1 structureare not observed compared with the toxin-freeapo-VSD1 structure (fig. S4E). Intriguingly, theAaH2-VSD1 complex does share some resem-blance to the AaH2-VSD4–deactivated state struc-ture, particularly along the S3-S4 loop interface(Fig. 5C).AaH2 targets only the S3-S4 loop and Tyr166-

NavPaS (S2 helix) to bury a limited surface area(~432 Å2) upon binding to VSD1 (Fig. 5, A andD).The NC domain of AaH2 remains unengaged atthe VSD1 receptor site, and consequently, Arg62

and His64 do not form securing interactions tothe DII PM (Fig. 5A). The lack of a robust bindinginterface between AaH2 and the compact DIIPM structure may rationalize the evolution ofa-scorpion toxins to more proficiently target Navchannels at neurotoxin receptor site 3. Beyondthis speculation, whether AaH2 can lock VSD1into an activated or deactivated state remainsunknown, but AaH2 may lack sufficient multi-point contacts to impose voltage-sensor trappingat the VSD1 receptor site (Fig. 5, C and D).To investigate the potential relevance of the

VSD1 receptor site, because NavPaS remains re-calcitrant to functional recordings (10, 68), weturned to the sequence-relatedBlatella germanicacockroach BgNav1 channel. Exemplar S3-S4 loopmutations (45) expected to perturb AaH2 bindingat VSD1 and VSD4were introduced in the BgNav1channel and assayed in Xenopus oocytes (Figs. 3,D and G, and 5D). AaH2 (100 nM) inhibited fastinactivation of WT-BgNav1 and VSD1-Asp220Arg-BgNav1 channels in a similar manner, but theVSD4-Asp1670Arg-BgNav1 mutation eliminatedthe effect (fig. S8, A to D). These results alignwith the expected action of a-scorpion toxins onNav channels and demonstrate that an offendingmutation can abolish the VSD4 interaction.Examined at 500 nM AaH2, a slow-onset

effect of current inhibition was observed in theWT-BgNav1 channel, in addition to inhibition offast inactivation (Fig. 5E and fig. S8, E and F).The VSD4-Asp1670Arg-BgNav1 mutation elimina-ted the toxin effect on fast inactivation, but theslow onset of current inhibition was still presentat 500 nM AaH2 (Fig. 5E). Notably, the VSD1-Asp220Arg-BgNav1 mutation showed the opposite

effect: Fast inactivation was inhibited, but theslow onset of current inhibition was abolishedat 500 nM AaH2 (Fig. 5E). When the test pulsefrequency was increased from 0.2 to 1 Hz, theonset of current inhibition was faster in WT-BgNav1 and VSD4-Asp1670Arg-BgNav1 channelsat 500 nM AaH2 (Fig. 5E), suggesting a toxininteraction with an activated state. These phys-iological data substantiate the biophysical rel-evance of the AaH2 interactions observed in thecryo-EM structure, including the unanticipatedAaH2-VSD1 receptor site complex.

DIV gating charges couple to the CTD

Fast inactivation is a hallmark of Nav channelfunction, but the structural basis of this processhas remained uncertain. In the AaH2-VSD4-NavPaS channel structure, AaH2 has trapped

VSD4 in a deactivated conformation (Figs. 2Band 6A), providing a new template to evaluatethe potential electromechanical coupling mech-anismof fast inactivation inNav channels (Movie 3).In theAaH2-VSD4–deactivated state, theR5 gatingcharge joins with K7 and R8 from the S4-S5 linkerto form an electrostatic bridge to conserved acidicresidues on the a1 helix of the CTD (Fig. 6A andfig. S6). We refer to this unpredicted molecularinterface as “switch 1.” If this electrostatic bridgerepresents a physiologically relevant interaction,then a simplistic two-step structuralmodel for thefast-inactivation mechanism emerges (Fig. 6, Ato C, and Movie 3). We first must hypothesizethat the electrostatic bridge physically restrainsthe CTD, which in turn promotes binding of theDIII-DIV linker across the CTD and DIV-S6. Wetentatively refer to these DIII-DIV linker inter-actions as the electrostatic latch, or “switch 2.”During step 1, VSD4 activation severs the electro-static bridge (switch 1) to release a key physicalconstraint on the CTD (Fig. 6, A, B, and D), likelystraining the DIII-DIV linker interactions (Fig.6E). During step 2, increased positional dynamicsof the CTD will release the electrostatic latch(switch 2), allowing the fast-inactivation gatingparticle (i.e., IFM motif) to bind the PM and ter-minate Na+ conductance (Fig. 6, B, C, and E).

Our AaH2-boundVSD4-NavPaS channel struc-ture suggests that the intracellular DIV gatingcharges are connected to the CTD throughelectrostatic bridging interactions in a restingstate (Fig. 6, A and D). To assess a link betweenthis putative electrostatic bridge (switch 1) andfast inactivation, we generated mutations of con-served acidic CTD residues in the related BgNav1channel (fig. S6) and indeed observed enhancedsteady-state inactivation (SSI) and acceleratedfast inactivation, among other phenotypes (fig. S9,A to C, and table S2). We next tested the func-tional role of this potential switch 1 interface inthe human Nav1.5 cardiac channel, noting thatlong QT and Brugada syndrome mutations arecommonly found in this region (Fig. 6, F and G)(69, 70). Charge reversal of conserved acidic CTDresidues to lysine (K) (CTD-3Kmutant: Asp1789Lys,Asp1792Lys, and Glu1796Lys) produced an ~8-mVleft shift in themidpoint of SSI and an acceleratedtime constant for fast inactivation (Fig. 6F, fig.S9D, and table S2), suggesting that breaking theelectrostatic bridge is associated with entry intothe fast-inactivated state. Conversely, reversal ofthe K7 andR8 gating charges to glutamic acid (E)(KR-2E mutant: Lys1641Glu and Arg1644Glu) alsocaused a left shift in SSI, with a shallowed slopeexpected for such severe gating chargemanipula-tion (Fig. 6F, fig. S9D, and table S2). Notably,when the gating charge and CTDmutationswerecombined into a single Nav1.5 construct (2E+3Kmutant), we observed that SSI was right-shiftedin this “charge-swapped”mutant channel relativeto either single alteration alone (Fig. 6F, fig. S9D,and table S2). This result supports a functionalcoupling interface across the electrostatic bridginginteractions observed within the AaH2-VSD4-NavPaS channel structure (Fig. 6, A and D). Toexplore the generality of these observations fur-ther, an analogous CTD-2K mutation (Glu1773Lysand Glu1776Lys) and R8D (i.e., R8→D) gatingcharge reversal were examined in the humanNav1.7 channel and found to produce similarphenotypes (fig. S9, F and G, and table S2). In-terestingly, a subtle depolarizing shift in themidpoint for activation was commonly observed(fig. S9, A, E, and G, and table S2), which mightarise as a result of accelerated fast inactivation(17, 71, 72). Overall, our physiological data supportamodel in whichDIVgating charges participate ina state-dependent interface with the acidic CTDand that these electrostatic interactions appre-ciably modulate Nav channel activation and fast-inactivation properties.

Discussion

We used single-particle cryo-EM methods to vi-sualize the a-scorpion toxin AaH2 in complexwith a chimeric Nav1.7 VSD4-NavPaS channel.AaH2 traps VSD4 in a deactivated state by wedg-ing into the extracellular cleft to formmultipointcontacts across neurotoxin receptor site 3. Arg62

and His64 of AaH2 target a constellation of resi-dues on theDI PM that help guide the toxin into astable binding pose (Fig. 3B). Mutagenesis studiesand a neutralizing monoclonal antibody confirman important role for these basic residues in


Movie 3. A potential structural model of fastinactivation in Nav channels. Trajectory(morph) between the AaH2-bound and apo-VSD4-NavPaS channel structures combinedwith the human Nav1.4 channel structure (PDB6AGF). AaH2 and b subunits are omitted forclarity, and the CTD in Nav1.4 was not modeledor depicted here.


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A

D

S4“down”

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CTD CTD CTD disordered

E

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E1784

Fig. 6. Proposed structural model of fast-inactivation gating inNav channels. (A) Side view of the AaH2-VSD4-NavPaS complex structurewith R1 to R8 gating charges shown in blue, conserved acidic switch 1(CTD) and switch 2 (DIV S6) residues in red, DIII-DIV switch 2 residuesin cyan, and the IFM-like motif residues in pink. AaH2 is omitted forclarity. (B) Side view of the apo-VSD4-NavPaS structure with residueshighlighted as in (A). (C) Side view of the human Nav1.4 channel cryo-EMstructure (PDB 6AGF) is shown with residues highlighted as in (A). TheCTD appears disordered in this structure, and the b1 subunit is omitted forclarity. (D) Close-up view of the switch 1 interactions, comparing theVSD4-deactivated and apo-VSD4–activated state structures. (E) Close-upview of the switch 2 interactions, comparing the apo-VSD4–activatedand human Nav1.4 channel structures. (F) Electrophysiological character-ization of human Nav1.5. Shown at the top are example traces of WT and

mutant Nav1.5 channels expressed in Xenopus oocytes. Oocytes were heldat −120 mV and pulsed from −80 to 40 mV for 30 ms. The graph on thebottom left shows steady-state inactivation as a function of voltage, whereoocytes were subjected to a 500-ms conditioning pulse at the indicatedvoltage, followed by a 1-ms step to −100 mV and a 20-ms test pulseat −20 mV. The graph on the bottom right shows the rate (t) of fastinactivation from single exponential fits of Nav activation in response todepolarization using the voltage protocol described above. Error barsrepresent mean ± SEM. (G) Structural model of human Nav1.5 based onthe AaH2-VSD4-NavPaS channel complex, showing switch 1 and switch2 residues (sticks) and side chains conserved in human Nav channelsthat are mutated in disease (spheres). Side chains of pathogenicmutations discussed in the text are indicated (R5, R8, Lys1505, and Glu1784).S1N, pre-S1 helix.


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Nav channel modulation (Fig. 3, F to H) (63).Comparison to an apo-VSD4-NavPaS channelstructure establishes that the S4 helix and S3-S4loop undergo substantial conformational changesduring VSD4 activation (Fig. 2, A to E). Con-sequently, AaH2 sterically occludes VSD4 activa-tion by forming a number of interactions thatserve to pin the S3-S4 loop and S4 helix into adeactivated conformation (Figs. 3, A to D, and4F). These AaH2-mediated interactions ration-alize the high potency and state dependence ofNav channel modulation (Fig. 1C).The long-chain a-scorpion toxin AaH2 is a

proficient gatingmodifier of Nav channels. AaH2modulates multiple Nav channel subtypes de-spite contacting VSD4 residues that are selectivitydeterminants for an emerging class of small-molecule antagonists (Figs. 1B and 5E, fig. S6,andMovie 1) (50–52, 54). AaH2 embraces a highlyconserved PM glycan on the VSD4-NavPaS chan-nel (Fig. 3B), which may in part explain the in-creased potency of AaH2 for Nav1.2 over Nav1.7(Fig. 1B and fig. S6). A previously unknownactivated-VSD1 receptor site highlights the in-trinsic affinity of AaH2 for Nav channel VSDs(Fig. 5, A and B), where AaH2 appears competentto affect BgNav1 channel gating at increasedtoxin concentrations (Fig. 5E). By contrast, AaH2can potently and robustly antagonize VSD4 bytrapping a deactivated state (Figs. 3G and 4, Dto F), underscoring the state dependence re-quired by a-scorpion toxins to modulate fastinactivation. Mechanistically, AaH2 is distinctfrom other structurally characterized gatingmodi-fiers like GX-936 and Dc1a, which directly contactS4 gating charges to trap activated conformationsof their VSD targets (fig. S10, A to C) (52, 68). Themultipoint contacts made by AaH2 across VSD4,however, are reminiscent of the transmembrane-linking interactions used by divalent cations toantagonize VSD activation in TPC1 andHv1 chan-nels (fig. S10, D and E) (73–75). The details ofAaH2 action and polypharmacology uncoveredhere provide insight into a-scorpion toxin evolu-tion and clues to design new Nav channel mod-ulators, although understanding the influence ofb subunits will require further study (fig. S10F)(5, 76, 77).The structural basis of voltage sensing and

electromechanical coupling in Nav channels thatleads to fast inactivation has remained enigmaticowing to technical challenges. Here, our use of apotent a-scorpion toxin has helped to elucidatethe VSD4 activation pathway (Movie 2). The S4helix translates ~13 Å with two gating charges(R3 and R4) transported across the central HCS(Fig. 2, A to D), consistent with prior electro-physiological measurements (14, 18, 25, 52). Theoverall translation of the S4 can be described as asliding helix, although the S4 itself remains in a310-helical conformation across the HCS, and theS1 to S3 core region undergoes a notable rigidbody displacement (fig. S11A). Studies have in-dicated that a-scorpion toxins do not trap a trueresting state of VSD4 (18), raising the possibilitythat we have visualized a potential deactivatedintermediate. Still, in the AaH2-VSD4–deactivated

state structure, the R5, K7, and R8 gating chargesform a dense network of electrostatic interactionswith acidic residues on the proximal surface ofthe intracellular CTD (Fig. 6, A and D). Thesepreviously unseen state-dependent bridging in-teractions may represent a view of the elusiveDIV-coupling interface to the fast inactivationgating machinery.Although it is tenuous to assign functional

states to Nav channel structures (fig. S11, B andC), we postulate that the available eukaryotic Navchannel structures outline a provisional two-stepstructural model of the fast-inactivation mecha-nism, as summarized in Fig. 6, A to E, andMovie 3.We propose that the AaH2-VSD4–deactivatedstate structure represents an early resting state ofthe fast-inactivation machinery (Fig. 6, A and D).Here, the R5, K7, and R8 gating charges formstate-dependent electrostatic bridging (switch 1)interactions with acidic residues on the a1 helixof the CTD, while basic residues from the DIII-DIV linker form electrostatic latch (switch 2)interactions with acidic residues on the CTD andDIV-S6helix (Fig. 6, A andE, and fig. S6). In step 1,voltage-dependent VSD4 activation will severswitch 1 interactions to release a key physicalconstraint on theCTD, producing an intermediatestate represented by the apo-VSD4-NavPaS chan-nel structure (Fig. 6, A, B, and D). In step 2,VSD4-releasing of the CTD will lead to increasedstrain on switch 2 interactions until the IFMmotif is liberated to find its PM-receptor site,as seen in the human Nav1.4 channel structure(Fig. 6, B, C, and E) (12), which will ultimatelyterminate Na+ conductance. Why the NavPaSchannel has allowed us to capture a potentialgating intermediate remains unknown (Fig. 6B),but partial activation of VSD3 or other sequencedifferences might provide justification (figs. S6and S11B). Nevertheless, the NavPaS channelchassis serves to highlight the CTD as an electro-negative nexus that may link VSD4 activationstatus to the fast-inactivation IFM-motif gatingparticle (Fig. 6, A, B, and D). The apo-VSD4-NavPaS andWT-NavPaS channel structures arehighly similar (10), as are the human and eelNav1.4 channel structures (11, 12), further sug-gesting that physiologically relevant states maybe depicted in Fig. 6 and Movie 3. Overall, wehave outlined a basic structural framework thatmay begin to describe molecular events occur-ring during fast inactivation in Nav channels.We recognize that our structure-based model

does not attempt to account for known differ-ences in fast-inactivation properties among Navchannel subtypes, nor the differential impact ofcell type or auxiliary subunits (e.g., b subunits or,calmodulin) (78–83). However, our simplisticfast-inactivation model does begin to rationalizeimportant literature on Nav channels. Hodgkinand Huxley first noted the slower “h” inactivationgating-particle (8), but the basis for this essentialfeature of electromechanical coupling in Navchannels has remained unknown. Here, the re-quirement for VSD4 gating charges (e.g., R5) tofirst break electrostatic bridging interactionswith the CTD (switch 1) may begin to account

for the slower activation kinetics of VSD4 rel-ative to VSD1 to VSD3 (Fig. 6, A, B, and D) (15, 17).Since Noda, Numa, and co-workers first clonedtheNav channel (9), it has also remainedunknownwhy DIV, uniquely among VSDs, contains eightgating charges (fig. S6) compared with the fourto six gating charges found in DI to DIII. Theadditional K7 and R8 gating charges of DIV arenow seen to participate in a dense network ofelectrostatic bridging contacts to the CTD (switch 1)(Fig. 6D), where these S4-S5 linker–CTD inter-actions may earmark DIV specialization duringeukaryotic Nav channel evolution.Numerous pathogenic mutations map to the

electrostatic bridge (switch 1) and electrostaticlatch (switch 2) regions defined by our fast-inactivation model (Fig. 6G), supporting thephysiological relevance of these interfaces. Anepilepsy syndrome (GEFS+) mutation in Nav1.1targeting R5 (Arg1648His) predicted to weakenswitch 1 interactions (Fig. 6G) produces persistentcurrent and accelerates recovery from fast in-activation (80, 84). Another GEFS+ mutation tar-geting R8 (Arg1657Cys) at the switch 1 interfaceproduces a depolarizing shift in activation andreduces channel current density (85). Long QTand Brugada syndrome mutations bombardswitch 1 and switch 2 regions in Nav1.5 (69),exemplified by the DIV-S6 helix Glu1784Lysmutation predicted to destabilize the switch 2interface (Fig. 6G). Glu1784Lys produces a hyper-polarizing shift in SSI and persistent current inNav1.5 (83, 86), and similar alterations have beenreported when acidic a1-helix residues along theCTD are neutralized (83). A detailed study ofGlu1784Lys demonstrated a hyperpolarizing shiftof VSD4 gating-charge movement, providing apotential integrated view of the switch 1 andswitch 2 coupling interfaces defined by our fast-inactivation model (71). Mutations that perturbthe DIII-DIV linker–CTD interactions also causedisease (Fig. 6G), including the DK1505PQ in-sult that produces persistent current in Nav1.5(69, 87) (Fig. 6G). Charge reversal or neutraliza-tion of Lys1505 in Nav1.5 and Nav1.3 producessimilar effects on fast inactivation (78, 88),whereas phosphorylation of Ser1506 by proteinkinase C slows inactivation in Nav1.2 (89) andcauses a hyperpolarizing shift of SSI in Nav1.5(90). Although the phenotypes and magnitudeof alterations may depend on the Nav channelsubtype or auxiliary subunits present, the per-turbations that we observe with targeted switch1 mutations in the BgNav1, Nav1.5, and Nav1.7channels (Fig. 6F; fig. S9, A to G; and table S2)overlap with the defects of disease-causingmutations identified in human patients.This study details the first high-resolution snap-

shots of a-scorpion toxin action on Nav channels,including pharmacological trapping of VSD4in a deactivated state, and the resolution of anunpredicted VSD1 receptor site. When combinedwith other recent eukaryotic Nav channel struc-tures, our results outline a structural frameworkto understand the mechanisms of voltage sens-ing, electromechanical coupling, and fast inactiva-tion operating inNav channels.We have proposed



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a simplistic fast-inactivation model that shedslight on the functional and evolutionary spe-cialization of DIV and also advances our under-standing of many pathogenic Nav channelmutations. Overall, our study provides templatesfor the design of new Nav channel modulatorsand a foundation to demystify the structuralbasis of fast inactivation.

Materials and methodsExpression and purification of chimericand wild-type NavPaS channels

Similar to the original report (10), an optimizedcodingDNA forNavPaS andNav1.7 VSD4-NavPaSchimeras containing a StrepII and FLAG tag intandem at the N terminus was cloned into a pRKvector behind a CMV promoter. HEK293 cells insuspension were cultured in SMM 293T-I me-dium under 5% CO2 at 37°C and transfectedusing PEI when the cell density reached 4 × 106

cells per ml. Transfected cells were cultured for48 hours before harvesting.Fifty grams of harvested cell pellet was

resuspended in 250 ml of 25 mM Tris pH 7.5,50 mM NaCl, 1 mg/ml benzonase, 1 mM PMSFand Roche protease inhibitor tablets. Cellswere lysed by gentle sonication, and NavPaSchannel proteins were subsequently solubilizedby addition of 1% digitonin supplemented with0.1% cholesteryl hemisuccinate (CHS) for 2 hoursat 4°C under gentle agitation. Insoluble debriswas pelleted by ultracentrifugation at 40,000 rpmfor 45 min, and the supernatant containing thesolubilized protein was collected for affinitypurification by batch-binding to 5 ml of M2-agarose FLAG resin (Sigma) for 1 hour at 4°C.Unbound proteins were washed with nine col-umn volumes (CV) of purification buffer [25 mMTris pH 7.5, 50 mM NaCl, 0.1% (wt/v) digitonin,0.01% CHS], followed by three CV supplementedwith 500 mM NaCl, and eluted with six CV ofpurification buffer supplementedwith 300 mg/mlFLAG peptide (Sigma). The eluent was collectedand applied to 3 ml Streptactin resin by gravity(six times). Unbound proteins were washed with10 CV of purification buffer and eluted with fiveCV of purification buffer supplemented with2.5 mM desthiobiotin. NavPaS proteins werethen concentrated with 100-kDa MWCO con-centrators to ~6 mg/ml and injected onto aSuperose 6 3.2/300 column attached to an AKTAsystem (GE Healthcare) for size-exclusion chro-matography into purification buffer.

Purification of AaH2

Native AaH2 was purified as previously described(91). Briefly, the Androctonus australis Hectorvenom (10 g) was extracted at 4°C with water,centrifuged 40 min, 13,000g at 4°C. The super-natant was adjusted with 0.1 M in ammoniumacetate, pH 8.5, and immediately filtered througha set of four Sephadex G-50 columns connectedin series. The fraction containing AaH2 was fur-ther submitted to cation exchange chromato-graphy onAmberlite CG-50 in 0.2M ammoniumacetate, pH 6.7. Quantification and homogeneityof the pure toxin was achieved by UV spectrum,

electrophoresis on 15% polyacrylamide gel innative conditions, amino acid analysis, and bymass spectrometry analysis (7244 Da). Standardbiological tests were performed to confirm bio-logical activity of the toxin.

Differential scanning fluorimetry

Melting experiments were conducted on a Pro-metheus NT48 (NanoTemper technologies) by mea-suring the tryptophan fluorescence 330/350 nmratio of protein samples concentrated at 0.3mg/mlin a standard capillary. AaH2 and GX-936 weremixed with purified NavPaS proteins 30 min priorto performing the experiment.

Cryo-EM sample preparation anddata acquisition

Native AaH2 was added to the VSD4-NavPaS chan-nel at 4:1 molar ratio prior to the size-exclusionstep at a final concentration of 200/50 mM. Gridswere prepared in the following manner. Holeycarbon grids (C-flat, R 2/1 200 meshCu; Protochips)were plasma etched using the Solarus plasmacleaner (Gatan) in the hydrogen-oxygen setting.Grids were etched for 4 min on each side toremove burrs from hole edges. The grids werethen coated on both sides with 5 nm of Au/Pd,which was plasma deposited using the LeicaACE600 (Leica). Three microliters of the peakfraction was applied to a glow-discharged holeyAu/Pd grid, incubated for 60 s, then hand blottedand another 3 ml of the peak fraction was ap-plied. Grids were then blotted in Vitribot Mark IV(Thermofisher) using 5-s blotting time with 100%humidity, and plunge-frozen in liquid ethanecooled by liquid nitrogen. Movie stacks werecollected using SerialEM (92) on a Titan Kriosoperated at 300 keV with bioquantum energyfilter equipped with a K2 Summit direct elec-tron detector camera (Gatan). Images wererecorded at 165000× magnification correspond-ing to 0.849 Å/pixel, using a 20-eV energy slit.Each image stack contains 40 frames recordedevery 0.25 s for an accumulated dose of ~40 e/Å2

and a total exposure time of 10 s. Images wererecorded with a set defocus range of 1 to 2.5 mm.

Cryo-EM data processing

Image stacks were processed using cisTEM(93). Frame motion was corrected and contrast-transfer function (CTF) parameters were fit frommovies using the 30-4 Å band of the spectrum.Images were resampled to 1 Å per pixel, andthose with CTF fits to 5 Å or better were selectedfor particle picking using a soft-edge disc as atemplate (4 s peak threshold, with maximumand characteristic radii of 90 and 65 Å, respec-tively). Three rounds of 2D classification with100 classes and a box size of 250 pixels wereperformed to sort AaH2-VSD4-NavPaS proteinparticles from debris and other false positives.The remaining, 504,252 particles were subjectedto a global angular search and classification intofour classes, using the published NavPaS EMmap (EMD-6698) low-pass filtered to 40 Å asa reference volume. Three classes, containing433,112 particles, were selected for a local angular

search and classification into three classes withthe best output from the previous global searchas a template. One class with the best structuralfeatures containing 251,714 particles was selectedfor 3D refinement. To avoid overfitting of the de-tergent micelle, a protein-only mask was createdfrom the published NavPaS model and used tolow-pass filter nonprotein regions of the map at20 Å between refinement iterations (94). Iterativeglobal and local refinement improved the pro-teinaceous features of the map, at which pointa newmask was created from the latest map, fora final refinement run using frequencies up to1/5.0 Å−1 and yielding a map at 3.5-Å resolution(FSC = 0.143). For model building and figurepreparation, maps were sharpened in cisTEMby (i) applying a B-factor of −90 Å2 from theorigin of reciprocal space to 1/10 Å−1, (ii) flatteningtheir rotational power spectrum from 1/10 Å−1

onwards, (iii) applying a B-factor of −30 Å2 inthat same range, and (iv) applying a figure-of-merit filter (95). Maps were subsequently filteredaccording to the local resolution, using reimple-mentation of blocres (96) within cisTEM and amodified resolution criterion (Rohou, in prep-aration). The same workflow was followed forthe apo-VSD4-NavPaS dataset, and relevant num-bers of particles can be found in fig. S2.

Model building

The structure of WT-NavPaS (PDB 5X0M) wasused as a template for modeling the Nav1.7-VSD4-NavPaS coordinates in SwissMODEL (97).The resulting model and the high-resolutioncrystal structure of AaH2 (PDB 1PTX)were fit asrigid bodies into the cryo-EMmap. After manualadjustments to the channel-toxin model, a singleround of real space refinement using the phenix.real_space_refinement tool with tight secondarystructure restraints (98) was used to correct globalstructural differences between the initial modeland the map. The model was further manuallyadjusted in Coot (99) through iterative roundsof model building and real space refinement.Phenix.mtriage was used to compute model vsmap FSC curves (fig. S2) that are consistent withour resolution estimate from the half-map FSCmeasurement.A definitive binding pose for the VSD1-bound

AaH2 could be determined, although density formany side chains does not allow for unambiguousassignment. Several attempts at 3D classification,including using approaches focused on the VSD1binding site, did not yield improved resolution orseparation between particles with bound andunbound AaH2 on VSD1. Our confidence in theAaH2-VSD1 complex is supported by the observa-tions that (i) the NavPaS channel is highly asym-metric, which makes it unlikely there werefrequent misalignments by 90° around thepore axis, and (ii) no such extra densities canbe seen for other major features protrudingfrom the membrane, e.g., the extended DI S5-S6turret loop.For the apo-VSD4-NavPaS channel structure,

the model was built and refined similarly asdescribed above.



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Xenopus oocyte recordings (BgNav1)All Blattella germanica Nav (BgNav1) mutantswere generated using the Gibson assemblymethod. For each mutant, four linear DNA frag-ments with approximately 20-bp homology armswere generated using PCR (PhusionHigh-FidelityDNA Polymerase, New England Biolabs, USA).Fragments were purified (MinElute PCR Puri-fication Kit, Qiagen, USA) and subsequently in-cubated for 2 hours at 55°CwithGibsonAssemblyMaster Mix (New England Biolabs, USA). “Assem-bled”plasmidswere then transformed (CopyCutterEPI400, Epicentre, USA) and mutagenesis wasconfirmed by automated Sanger sequencing.cRNA was synthesized using T7 polymerase(mMessage mMachine kit, Life Technologies,USA) after linearizing the DNA with appropriaterestriction enzymes.BgNav1 control and mutant constructs were

expressed inXenopus laevis oocytes (sourced fromXenopus one, USA), and electrophysiologicalrecordings were taken after 2 to 4 days post cRNAinjection. Oocytes were incubated at 17°C inBarth’s medium (96 mMNaCl, 2 mM KCl, 5 mMHEPES, 1 mMMgCl2, and 1.8mMCaCl2, 50 mg/mlgentamycin, pH 7.6 with NaOH) and studiedusing the two-electrode voltage-clamp recordingtechnique (OC-725C, Warner Instruments) witha 150-ml recording chamber. All datawere filteredat 4 kHz and digitized at 20 kHz using pClamp10 software (Molecular Devices, USA). The exter-nal recording solution used was ND-100 (100mMNaCl, 5 mM HEPES, 1 mM MgCl2, and 1.8 mMCaCl2, pH 7.6 with NaOH) and microelectroderesistances were 0.5 to 1.0 MWwhen filled with3 M KCl. All experiments were performed atroom temperature (~22°C), and AaH2 sampleswere diluted in ND-100 with 0.1% BSA. Leak andbackground conductances were identified andsubtracted by blocking the channel with TTX(Alomone Labs, Israel). After adding toxin to therecording chamber, equilibrationbetween channeland toxin wasmonitored using weak depolariza-tions (~20) at 5-s intervals unless otherwisenoted. Typically, voltage-activation relationshipswere recorded before and after toxin addition.Off-linedata analysiswasperformedusingClampfit10 (Molecular Devices, USA), Microsoft Excel(Microsoft, USA) and Prism 7 (GraphPad, USA).For data presented in figs. S8 and S9, significanceof all normalized conductance-voltage (G-V) andsteady-state inactivation (I-V) relationships wasanalyzed using two-way analysis of variance(ANOVA) with post hoc Tukey’s test. Individualtime point values for fast-inactivation time con-stants (t), persistent current, peak current, andrecovery from fast inactivation (RFI) were ana-lyzed using Student’s t test. Values in all casesreflect the mean, and error bars reflect SEM, P <0.05 (*) or 0.001 (**). For all data presented intable S2, Student’s t (unpaired) against wild-typewas used.

Xenopus oocyte recordings(human Nav1.5)

Mutations in thewild-type humanNav1.5 gene inthe pcDNA 3.1 vector were introduced via Gibson

assembly in conjunction with gblock synthesis(IDT, Coralville Iowa) and verified with auto-mated Sanger sequencing. In vitro transcriptionof cRNA was accomplished with the mMessagemMachine T7 ultra kit, following the manufac-turer’s instructions.For two-electrode voltage-clamp recordings,

RNA encoding wild-type and mutant Nav1.5channels were injected (50 to 100 ng) into Xenopusoocytes purchased from Ecocyte, Inc. (Austin,TX). Two-electrode voltage-clamp recordings wereperformed 1 to 3 days postinjection in oocyteRinger’s solution containing (in mM): 116 NaCl,2 KCl, 1.8 CaCl2, 2 MgCl2, 5 HEPES, pH 7.4. Elec-trodes had resistances of 0.3 to 0.6 MW whenfilled with 3 M KCl. The NPI Turbo-TEC 03X am-plifier was used. Signals were filtered at 20 kHzby the amplifier and digitized at 100 kHz bypClamp software (Molecular Devices, San Jose,CA, USA). Traces were filtered at 10 kHz for dis-play in figures. Where indicated, we co-injectedcRNA encoding the b1 with Nav1.5 at a 2:1 massratio of alpha to b. Although neither the activa-tion nor the rate of inactivation of Nav1.5 areaffected by coexpression of the b1 subunit withNav1.5 in Xenopus oocytes (100, 101), in our hands,co-injection with b1 caused a small (~3 mV) de-polarizing shift in steady-state inactivation thatwas consistent among variants and did not af-fect their relationship relative to one another(table S2). Offline analysis was performed usingClampfit 10 (Molecular Devices, San Jose, CA,USA) for peak current quantification and OriginPro (Northampton, MA) for fitting.

Whole-cell patch-clamp recordings(human Nav1.2 and Nav1.7)

Wild-type Nav1.7 voltage-clamp recordings wereobtained from HEK293 cells constitutively ex-pressing human Nav1.7 (GenBank Accession:NM_002977) and the human b2 subunit. HEK-TetOn cells were transfected with 2 mg of mutantNav1.7 DNA in the pBi vector containing thehuman b2 subunit using Lipofectamine LTX(Invitrogen) 15 to 20 hours prior to recording.Wild-type Nav1.2 voltage-clamp recordings wereobtained fromHEK293 cells heterologously express-inghumanNav1.2 (GenBankAcession:NM_021007)and the human b1 subunit. Cells expressingNav1.2were induced 15 to 20 hours prior to recordingusing doxycyclin.Whole-cell patch-clamp record-ings were obtained using aMolecular DevicesAxopatch 200B patch-clamp amplifier. The rec-ording pipette intracellular solution contained(in mM): 140 CsF, 10 NaCl, 1.5 MgCl2, 10 HEPES,5 EGTA adjusted to pH 7.3 with CsOH, os-molarity 300. The extracellular recording so-lution contained (in mM): 80 NaCl, 60 NMDG,4 KCl, 2 CaCl2, 1 MgCl2, 5 glucose, 10 HEPES,and containing 0.1% BSA (pH 7.4, osmolarity300 mOsm).Lyophilized powder of both native and wild-

type and toxin mutants (supplied by SmartoxBiotechnology) were resuspended in extracellularrecording solution, and the concentration checkedwith A280. Currents were recorded at 20-kHzsampling frequency and filtered at 5 kHz. Series

resistance compensation was applied at >80%.A stable baseline was established prior to toxinperfusion, after which increasing concentrationsof toxin were perfused using the Dynaflow Re-solve System (Fluicell) and resulting currents weremeasured after equilibrium reached. Data wasextracted using Clampfit (Molecular Devices),analyzed using the tidyverse package in Rstudio(HadleyWickham), andplotted inOrigin (OriginLabCorporation). A 15-ms window after channel open-ing was used to derive dose responses, the processof which is pictorially demonstrated in fig. S7A.EC50 measurements on Nav1.2 and Nav1.7 weremade while holding the membrane voltage at−100 mV. A 100-ms pulse to 0 mV was used toopen the channel. Using this protocol at a 0.5-Hzpulse rate, themembrane voltage ismaintained at−100 mV 95% of the time. For state-dependenceexperiments, the membrane voltage was heldat either −40 or −120 mV. A 20-ms prepulse to−150mVwas used to partially recover channelsfrom inactivation, followed by a 20-ms pulse to0 mV to open the channel. Using this protocol ata 0.2-Hz pulse rate, the membrane voltage ismaintained at −40 or−120mV99.2% of the time.

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http://www.ncbi.nlm.nih.gov/pubmed/16382098

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http://dx.doi.org/10.1085/jgp.62.4.375



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http://dx.doi.org/10.1016/j.toxicon.2006.09.017





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http://dx.doi.org/10.1038/320188a0


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ACKNOWLEDGMENTS

We thank our Genentech colleagues in the Neuroscience,Structural Biology, BioMolecular Resources, Early Discovery

Biochemistry, and Early Stage Cell Culture departments for theirsupport of this project and, in particular, A. Estevez, H. Xu,M. Dourado, L. Deng, J. Fuhrmann, C. Koth, and S. Hymowitz.We thank our colleagues at Smartox Biotechnology for generatingthe AaH2 derivatives used in this study. C.A.A. acknowledgesR. Kass for the wild-type Nav1.5 clone, and F.B. acknowledgesK. Dong for the wild-type BgNav1 clone. Funding: A.C. wassupported by an MRC Industrial iCASE Ph.D. studentship award(MR/N017927/1). D.T.I. was supported by the Cystic FibrosisFoundation (INFIEL17F0). J.P.L. was supported by theJames H. Gilliam Fellowships for Advance Study program throughthe Howard Hughes Medical Institute. M.-F.M.-E. acknowledgessupport from the Institut national de la santé et de la recherchemédicale (Inserm). P.E.B. was supported by the Centre national dela recherche scientifique (CNRS: PEPS-2009/PAGAIE). C.A.A. wassupported by GM122420 and is an American Heart Associationestablished investigator (5EIA22180002). F.B. was supported bythe National Institute of Neurological Disorders and Strokeof the National Institutes of Health (R01 NS091352). Authorcontributions: T.C. established conditions for protein purificationand toxin complex formation. T.C. and C.P.A. optimized cryo-EMsamples and, with assistance from C.C., collected cryo-EM data.T.C. and A.R. processed and determined cryo-EM structuresand, with J.P., performed structural analyses. Z.R.L. and Y.J.generated essential molecular biology reagents. M.-F.M.-E. andP.E.B. performed native AaH2 purifications and providedguidance on toxin pharmacology. A.C. and D.H.H. performedelectrophysiological analyses on human Nav1.2 and Nav1.7channels. D.T.I. and C.A.A. performed electrophysiological analyseson the human Nav1.5 channel. J.P.L. and F.B. performedelectrophysiological analyses on the cockroach BgNav1 channel.T.C. and J.P. prepared the manuscript with assistance andinput from all other authors. C.A.A., F.B., D.H.H., A.R., and J.P.are senior co-authors, and J.P. supervised the project.Competing interests: T.C., A.C., C.P.A., Z.R.L., Y.J., C.C., D.H.H.,A.R., and J.P. were all employees of Genentech, Inc., at the timeof this study; all other authors declare no competing financialinterests. Data and materials availability: Materials will be madeavailable upon request and material transfer agreement withGenentech or the appropriate party. Accession numbers for theapo-VSD4-NavPaS (VSD4-activated) cryo-EM model and maps areProtein Data Bank (PDB) 6NT3 and EMD-0500, respectively;accession numbers for the AaH2-VSD4-NavPaS (toxin-bound,VSD4-deactivated) cryo-EM model and maps are PDB 6NT4 andEMD-0501, respectively.

SUPPLEMENTARY MATERIALS

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channelsv-scorpion toxin action on NaαStructural basis of

Hackos, Alexis Rohou and Jian PayandehJian, Marie-France Martin-Eauclaire, Pierre E. Bougis, Claudio Ciferri, Christopher A. Ahern, Frank Bosmans, David H. Thomas Clairfeuille, Alexander Cloake, Daniel T. Infield, José P. Llongueras, Christopher P. Arthur, Zhong Rong Li, Yuwen

originally published online February 7, 2019DOI: 10.1126/science.aav8573 (6433), eaav8573.363Science

, this issue p. eaav8573; see also p. 1278Sciencelead to fast inactivation.interactions between VSD4 and the carboxyl-terminal region change as VSD4 activates and suggests how this wouldChowdhury and Chanda). The toxin traps VSD4 in a deactivated state. Comparison with the apo structure shows how

1.7, both in the apo state and bound to a scorpion toxin that impedes fast activation (see the Perspective byvhuman NaPaS is replaced with VSD4 fromvdetermined the structures of a chimera in which VSD4 of the cockroach channel Na

et al.four voltage-sensing domains (VSDs), with VSD4 playing an important role in fast inactivation. Clairfeuille inactivation, and mutants that impede this cause conditions such as epilepsy and pain syndromes. The channels have

) channels are key players in electrical signaling. Central to their function is fastvVoltage-gated sodium (NaHow activation leads to gating

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REFERENCES

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