Research Article

Structures of human Nav1.7 channel in complex with auxiliary subunits and animal toxins

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Science  22 Mar 2019:
Vol. 363, Issue 6433, pp. 1303-1308
DOI: 10.1126/science.aaw2493

Targeting sodium channels

Voltage-gated sodium (Nav) channels have been implicated in cardiac and neurological disorders. There are many subtypes of these channels, making it challenging to develop specific therapeutics. A core α subunit is sufficient for voltage sensing and ion conductance, but function is modulated by β subunits and by natural toxins that can either act as pore blockers or gating modifiers (see the Perspective by Chowdhury and Chanda). Shen et al. present the structures of Nav1.7 in complex with both β1 and β2 subunits and with animal toxins. Pan et al. present the structure of Nav1.2 bound to β2 and a toxic peptide, the µ-conotoxin KIIIA. The structure shows why KIIIA is specific for Nav1.2. These and other recently determined Nav structures provide a framework for targeted drug development.

Science, this issue p. 1303, p. 1309; see also p. 1278


Voltage-gated sodium channel Nav1.7 represents a promising target for pain relief. Here we report the cryo–electron microscopy structures of the human Nav1.7-β1-β2 complex bound to two combinations of pore blockers and gating modifier toxins (GMTs), tetrodotoxin with protoxin-II and saxitoxin with huwentoxin-IV, both determined at overall resolutions of 3.2 angstroms. The two structures are nearly identical except for minor shifts of voltage-sensing domain II (VSDII), whose S3-S4 linker accommodates the two GMTs in a similar manner. One additional protoxin-II sits on top of the S3-S4 linker in VSDIV. The structures may represent an inactivated state with all four VSDs “up” and the intracellular gate closed. The structures illuminate the path toward mechanistic understanding of the function and disease of Nav1.7 and establish the foundation for structure-aided development of analgesics.

Among the nine subtypes of human voltage-gated sodium (Nav) channels, Nav1.7, which is encoded by SCN9A and highly expressed in peripheral sensory neurons, has a direct association with pain syndromes (14). Mutations in Nav1.7 are found in many pain syndromes, including both extreme pain disorder and indifference to pain (table S1) (510). An accurate structural model of Nav1.7 would facilitate drug discovery for this promising target (1113).

Eukaryotic human Nav channels share high sequence similarity (fig. S1) (14, 15). Cryo–electron microscopy (cryo-EM) structures of representative Nav channels from insect, electric eel, and human reveal identical architecture of the core α subunit (1618). A single polypeptide chain, the α subunit, folds to four homologous repeats, each containing six transmembrane helices designated S1 to S6. The S1 to S4 segments in each repeat constitute the voltage-sensing domain (VSD) that attaches to the central ion-conducting pore domain (PD) enclosed by the S5 and S6 helices from the four repeats. The VSDs and PD segments conform to the canonical domain-swapped assembly that is prevalent in the voltage-gated ion channel superfamily (19, 20). The sequences between S5 and S6 comprise the selectivity filter (SF) that is sandwiched by two half-membrane-penetrating reentrant pore helices P1 and P2. Four distinct residues on the corresponding SF loci in the four repeats, Asp-Glu-Lys-Ala (DEKA), are the signature motif for Na+ selection (21) (fig. S1). Although the α subunit alone is sufficient for voltage-dependent gating of ion permeation, it is subject to regulation by one or more β auxiliary subunits (22). All four β subunits, β1 to β4, affect the channel properties of Nav1.7, although β1 and β2 are commonly coexpressed with the Nav1.7 α subunit for biophysical characterization (22, 23). Structures of the Nav1.4-β1 complex from both electric eel and human reveal the interaction details between α and β1 (17, 18), but the binding mode for other β subunits remains to be structurally elucidated.

Nav channels are targeted by various natural toxins and therapeutic drugs. These chemical compounds and peptides are generally classified into two classes, pore blockers exemplified by the small-molecule neurotoxins tetrodotoxin (TTX) and saxitoxin (STX) and peptidic gating modifier toxins (GMTs) that lock the channel in a particular functional state, hence altering the firing and propagation of action potentials (24, 25). Some toxins, particularly those with subtype specificity, provide leads for potential therapeutics for treatment of pain (26). The structures of a Nav channel from American cockroach, NavPaS, bound to TTX, STX, and Dc1a, a GMT from the venom of desert bush spider, provide a glimpse of the modulation of Nav channels by animal toxins (27).

Among the GMTs that target Nav channels, two tarantula toxins, protoxin-II (ProTx-II) and huwentoxin-IV (HWTX-IV), exhibit potent inhibition of Na+ current mediated by Nav1.7 (2832). Both toxins inhibit the activation of Nav channels by binding to the linker between the S3 and S4 segments (L3-4 loop) in the second VSD (VSDII), which is known as “site 4” (30, 33, 34). ProTx-II was also characterized to interact with VSDI and inhibit channel inactivation by binding to VSDIV in the activated channel (31, 35). Structural elucidation of the molecular details of toxin interaction with Nav1.7 may offer a path to potential drug discovery.

In this study, we report the structures of human Nav1.7 in the presence of both β1 and β2 subunits at resolutions of 3.2 Å determined using single-particle cryo-EM. Two combinations of toxins were supplemented, ProTx-II with TTX and HWTX-IV with STX. For simplicity, we will refer to these structures as Nav1.7-PT and Nav1.7-HS, respectively.


Structural determination of Nav1.7-PT and Nav1.7-HS

To simplify expression and purification of Nav1.7, we screened natural variants, each carrying a disease mutation (table S1). Out of 50 tested variants, Nav1.7 (Glu406→Lys, or E406K), a variant found in a patient with primary erythermalgia (36), expressed at least threefold higher than the wild-type channel. Whole-cell electrophysiological characterization of Nav1.7 in the presence of β1 and β2 subunits shows that Nav1.7 (E406K) exhibits a hyperpolarizing shift in activation (−10.6 mV) and prolonged fast-inactivation duration compared with the wild type (Fig. 1A, fig. S2, and table S2). These observations are consistent with the alteration of channel properties found for other mutants associated with primary erythermalgia (7, 3739).

Fig. 1 Structural determination of human Nav1.7 (E406K) in complex with auxiliary subunits and toxins.

(A) Electrophysiological characterization of wild-type Nav1.7 and the variant Nav1.7 (E406K) associated with primary erythermalgia in the presence of the β1 and β2 auxiliary subunits. See materials and methods, fig. S2, and table S2 for details. Nav1.7 (E406K) was used for structural determination. For simplicity, we will refer to this variant as Nav1.7 with regard to structural description. V, voltage; G, conductance; I, electric current; max, maximum value. (B) SEC purification of the human Nav1.7-β1-β2 complex. Mass spectrometric analysis (top) of the upper bands on the coomassie blue–stained SDS–polyacrylamide gel electrophoresis (bottom), indicated by the red arrow, confirmed the presence of all three subunits. The concentrated protein complex was supplemented with the toxin combinations of either ProTx-II with TTX (PT) or HWTX-IV with STX (HS) for cryo-sample preparation. UV, ultraviolet; mAU, milli–absorbance units. (C) Representative electron micrograph (top) and two-dimensional (2D) class averages (bottom). The green circles indicate representative particles in distinct orientations. The white scale bar in the bottom panel indicates 10 nm. (D) EM reconstructions of the human Nav1.7-β1-β2 in complex with PT (Nav1.7-PT) or HS (Nav1.7-HS), both at 3.2-Å resolution. The top panel shows the gold-standard Fourier shell correlation (FSC) curves for the 3D reconstruction of the two complexes. In the bottom panel, the local resolution map was calculated with Relion 2.1 and presented in Chimera.

Out of 16 liters of human embryonic kidney–293F (HEK-293F) cells cotransfected with plasmids for Strep and FLAG twin-tagged Nav1.7 and nontagged β1 and β2, about 100 to 200 μg of complex was obtained after tandem affinity purification and size exclusion chromatography (SEC) (Fig. 1B). The proteins purified in the buffer containing 0.04% (weight/volume) glyco-diosgenin (GDN, Anatrace) were further concentrated to about 2 mg/ml. A combination of ProTx-II (50 μM) and TTX (50 μM), or HWTX-IV (50 μM) and STX (11 μM), was separately added to the concentrated sample 30 min before making cryo grids.

Cryo-EM images were collected and processed following standard protocols (Fig. 1, B and C, and fig. S3A). From 263,205 and 275,630 selected particles, respectively, the structures of Nav1.7-PT and Nav1.7-HS were both determined at 3.2-Å resolutions (Fig. 1D; figs. S3B and S4; and table S3). For both complexes, the Nav1.7 core α subunit is well resolved. The extracellular immunoglobulin (Ig) domain and the single transmembrane helix (TM) of β1 are fully resolved, whereas only the Ig domain of β2 is discernible to a lower resolution of 4 to 5 Å (Fig. 1D). Two blobs of densities at moderate resolutions are found, respectively, above VSDII and VSDIV in Nav1.7-PT, and one adheres to the extracellular edge of VSDII in Nav1.7-HS. The difference between these two reconstructions and between them and our previous Nav1.4 map suggests that these additional densities belong to ProTx-II and HWTX-IV. However, the densities are at peripheral regions and were only resolved to moderate resolutions of ~5 Å, which does not allow accurate docking of the toxin structures (Fig. 2A and fig. S4A).

Fig. 2 Overall structures of Nav1.7-PT and Nav1.7-HS.

(A) One blob of density is above VSDII in Nav1.7-HS, and two are above VSDII and VSDIV in Nav1.7-PT. These densities belong to HWTX-IV and ProTx-II, respectively. The structures and the densities, which are low-pass filtered to 4.5 Å, are prepared in Chimera. (B) Overall structure of Nav1.7 in complex with β1 and β2. Nav1.7 is colored by repeats. The III-IV linker, which carries the fast-inactivation motif Ile-Phe-Met (IFM), is highlighted in orange. The sugar moieties are shown as black sticks, and the IFM residues are shown as spheres. All structure figures were prepared in PyMol (51) or Chimera.

For both structures, side chains could be assigned for residues 114 to 1768 except for the intracellular linkers I-II (residues 418 to 725) and II-III (residues 973 to 1174). Residues 20 to 192 were built for the β1 subunit, and the crystal structure of β2 (Protein Data Bank 5FEB) was docked into the density corresponding to β2-Ig with minor adjustment (Fig. 2B). Eleven glycosylation sites, six on Nav1.7, four on β1, and one on β2, were assigned (table S3). As most of the two structures containing Nav1.7-β1-β2 is identical, we will not distinguish the two complexes during structural illustration unless for specific discussions. Several minor structural shifts between Nav1.7-β1-β2 and Nav1.4-β1 (18) are described in the supplementary text and fig. S5.

Closed intracellular gate

In the structures of Nav1.4 from both electric eel and human, the intracellular gate is penetrated by an elongated detergent molecule, digitonin or GDN (17, 18). No such density is observed in the EM maps of the Nav1.7 complexes, although a similar concentration of GDN was used for purification and cryo-sample preparation of the Nav1.4 and Nav1.7 complexes. The S6 segments in repeats II to IV of Nav1.7 undergo slight counterclockwise twisting relative to those in Nav1.4 when viewed from the intracellular side (Fig. 3A). These seemingly minor changes result in the upward shift of the intracellular gate by ~5 Å, enclosed by four hydrophobic gating residues on the corresponding locus of each S6, Leu398-Leu964-Ile1453-Tyr1755, that are highly conserved among the Nav channels (Fig. 3, B and C, and fig. S1). Single point mutations of the corresponding residues of Leu398 and Leu964 are found in Nav1.5 in patients with long QT syndrome and Brugada syndrome, respectively (40, 41), whereas mutations of the Tyr1755-corresponding residues in Nav1.1 and Nav 1.5 are associated with seizure and long QT syndrome, respectively (42, 43).

Fig. 3

Closed intracellular gate. (A) The intracellular gate of Nav1.7 is slightly tightened compared with that of Nav1.4. Shown here and in (C) are intracellular views of the superimposed PDs of Nav1.7-HS (domain colored) and Nav1.4 (pale pink). The orange arrows indicate the slight rotation of the S6 segments in repeats II to IV of Nav1.7 relative to those in Nav1.4. (B) Two conformers of Tyr1755 in Nav1.7-HS result in the shift of the constriction site along the permeation path of Nav1.7. Shown in the left two panels are the respective permeation paths, calculated by HOLE (52), of Nav1.7-HS with Tyr1755 up (left, green) or down (right, purple). The corresponding pore radii of human Nav1.7 (green and purple) and Nav1.4 (gray) are compared in the right panel. The residues constituting the respective constriction sites in the two conformations of Nav1.7-HS are shown as sticks. Eα, extracellular α helix. (C) The intracellular gate of Nav1.7 is constituted by four hydrophobic residues on the corresponding locus of each S6, Leu398-Leu964-Ile1453-Tyr1755. The GDN molecule observed in Nav1.4, shown as pink thin sticks, cannot be accommodated by the closed gate in Nav1.7. Phe1755 of Nav1.7 in the type I (down) conformation is shown as ball and sticks. (D) Rotation of two aromatic rings closes the fenestration on the interface between repeats I and IV. Shown here is a side view of the superimposed PDs of Nav1.7 and Nav1.4. The type I and II conformers of Met1754 and Tyr1755 are shown as cyan and dark gray sticks, respectively, and the conformational shifts are indicated by orange arrows. The type II conformers of these two residues exhibit similar conformations to the corresponding ones in Nav1.4 (shown as thin pink sticks). The adjacent Phe387 and Phe391 on S6I (silver for Nav1.7) adopt distinct conformers from the corresponding ones in Nav1.4 (pink), leading to closure of the fenestration on this side wall of the pore domain. Conformational changes of the corresponding Phe residues from Nav1.4 to Nav1.7 are indicated by dark gray arrows. See fig. S6 for a detailed analysis. Single-letter abbreviations for the amino 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.

The local EM map reveals two distinct rotamers of Tyr1755 (Fig. 3D and fig. S6A). Although the main chain remains unchanged, the rotation of the aromatic ring results in two distinct conformers, which we refer to as type I and type II, for intracellular gating (Fig. 3, B and C). The type I Tyr1755 conformer is similar to the corresponding Tyr1593 in Nav1.4 (Fig. 3D, black sticks). The constriction with a diameter of ~4 Å is defined by Leu398 and Ile1453 (Fig. 3B, left). The narrowed gate can no longer accommodate GDN. In the type II conformer, the side chain of Try1755 rotates around Cβ by nearly 180°. Consequently, the aromatic ring directly cuts into the permeation path, completely sealing the intracellular gate (Fig. 3, B and C). The side chain of the preceding residue Met1754 also has two conformations. One is the same as that in Nav1.4, and the other, which we also refer to as the type II conformer, swings toward S6I (Fig. 3D and fig. S6A). The adjacent Phe387 and Phe391 on S6I both display distinct rotamers from those in Nav1.4 (Fig. 3D and fig. S6B). Such conformational shifts effectively avoid clashing with the type II conformer of Met1754, meanwhile leading to closure of the fenestration on the side wall constituted by repeats I and IV (Fig. 3D and fig. S6C).

Pore blockade by TTX and STX

TTX and STX have been used as potential therapeutics for treatment of pain (4446), and STX exhibits a lower affinity with Nav1.7 than with other subtypes (47). We recently reported the insect NavPaS in complex with TTX and STX (27). Given that not all the coordinating residues are invariant between NavPaS and Nav1.7, accurate structural models for Nav1.7 bound to these prototypal neurotoxins are necessary to facilitate future drug discovery. The resolutions of the central region are higher than the averaged 3.2 Å for both Nav1.7-PT and Nav1.7-HS, enabling reliable model building for TTX and STX and surrounding residues (fig. S7).

The binding sites and most interactions for TTX and STX are conserved between Nav1.7 and NavPaS (Fig. 4, A and B). The major distinction occurs in the coordination by repeat III. Two invariant residues in the other eight human Nav subtypes on the P2 helix, Met and Asp, are replaced by Thr1409 and Ile1410, respectively, in Nav1.7 (Fig. 4C). The hydroxyl group and main-chain amide of Thr1409 are both hydrogen bonded to the C10-OH of TTX, and the hydroxyl group of Thr1409 forms two hydrogen bonds with the carbamoyl group of STX (Fig. 5, A and B). In other subtypes, it is likely that the Asp corresponding to Ile1410 interacts directly with TTX and STX. The structure supports a previous analysis that variation from Met1409 and Asp1410 in other Nav channels to Thr and Ile, respectively, in Nav1.7 may account for the lowered affinity between Nav1.7 and STX (47). The molecular details for the recognition of TTX and STX by Nav1.7 may facilitate future optimization of ligands for pain relief.

Fig. 4

Pore blockade by TTX and STX. (A) Specific interactions between TTX and Nav1.7. Extracellular views of Nav1.7 (left) and the superimposed SF region of Nav1.7 and NavPaS (right) are shown. NavPaS is colored light purple. The coordination of TTX by Nav1.7 is nearly identical to that by NavPaS except for residue variations on P2III. The SF residues Asp361-Glu930-Lys1406-Ala1698 (DEKA) are shown as thick sticks and color labeled. The red circles indicate the difference in the coordination of the toxin by Nav1.7 and NavPaS. (B) Specific interactions between STX and Nav1.7. The conserved Met and Asp on helix P2 in the other eight human Nav subtypes, among which Asp is expected to participate in STX binding, are replaced by Thr1409 and Ile1410, respectively, in Nav1.7. The polar interactions are represented by red dashed lines. The red circles highlight Thr1409-Ile1410 and the corresponding residues in NavPaS. (C) Sequence alignment of the SF elements and P2 helices in each repeat of human Nav channels and NavPaS. The panel is adapted from our recently published sequence alignment, with minor adjustment (27). The residues whose side chains participate in TTX and STX coordination are indicated by gray and brown squares, respectively, below the alignment. The varied toxin-coordinating residues between Nav1.7 and other channels are in white text on a brown background.

Fig. 5 Binding sites and potential working mechanism for HWTX-IV and ProTx-II.

(A) Structural differences between Nav1.7-HS (color coded) and Nav1.7-PT (white). An extracellular view of the two superimposed structures is shown. β2-Ig domains are shown in semitransparent surface representation. The lack of specific interaction between β2-Ig and Nav1.7, other than the disulfide bond, may account for the poor resolutions and the flexible positioning of β2-Ig in the two reconstructions. VSDII, which provides the docking site for both GMTs, is highlighted by pink shading. (B) Slight conformational changes of VSDII between Nav1.7-HS and Nav1.7-PT. The S4 segment exists as a 310 helix in both structures. Note that the Cα atoms and the side groups of GC residues R2 to R4 in Nav1.7-PT move slightly toward the extracellular side relative to those in Nav1.7-HS. (C) HWTX-IV and ProTx-II bind to the similar site 4 on VSDII. In the superimposed overall structures of Nav1.7-HS and Nav1.7-PT, ProTx-II (orange) and HWTX-IV (light purple) are largely overlapped. The densities for the toxins are low-pass filtered to 4.5 Å in Chimera. (D) Distinct binding modes for ProTx-II by VSDII and VSDIV. When ProTx-II–loaded VSDII and VSDIV are superimposed, the difference in the position of the toxin is evident. See fig. S8 for a more detailed analysis on the binding of ProTx-II and HWTX-IV by Nav1.7. (E) Potential working mechanisms for GMTs. The cartoon is derived from the structures of NavPaS in complex with Dc1a (left) and Nav1.7 bound to GMTs (right). An entity other than a VSD, such as the PD and the surrounding lipid bilayer, provides the support to anchor the GMT, which then can lock the target VSD in a functional state through specific interactions. ECL, extracellular loop. The changing colors for GMT in the right panel indicate the amphiphilic nature of GMTs like HWTX-IV and ProTx-II.

Conformational differences of VSDII in the presence of ProTx-II and HWTX-IV

The structures of Nav1.7-PT and Nav1.7-HS are nearly identical except for overall shifts of the β2 subunit and local differences in VSDII (Fig. 5A). Cys895 on the extracellular loop of repeat II forms a disulfide bond with β2-Cys55 (fig. S1). Pivoting around this disulfide bond, the Ig domains in the two reconstructions deviate from each other by about 15° (Fig. 5A). The shift may reflect the overall flexibility of the immunoglobulin G (IgG) domain relative to Nav1.7 owing to the lack of specific interactions other than the disulfide bond.

The structural shifts of VSDII between Nav1.7-PT and Nav1.7-HS are mainly found in the S3 and S4 segments (Fig. 5B). The entire S4 segment in VSDII exists as a 310 helix in both reconstructions, but a detailed comparison of Nav1.7-PT and Nav1.7-HS shows slight motions between the two S4 segments. The last two gating charge (GC) residues, Arg844 (R5) and Lys847 (K6) can be well superimposed, but the segment deviates at Arg841 (R4) and above. The helical turns in Nav1.7-PT are slightly more elongated, shifting the Cα atoms of R2 to R4 toward the extracellular side relative to the corresponding ones in Nav1.7-HS. The movements of the side chains are more pronounced, with the guanidinium groups of R2 and R3 both above the conserved coordinating residue Asn780 (An1) in Nav1.7-PT, whereas only that of R2 is above An1 in Nav1.7-HS (Fig. 5B, right).

Considering the identical protein purification procedure, it is reasonable to attribute the structural shifts of VSDII to the association of ProTx-II and HWTX-IV (Fig. 5, C and D, and fig. S8). The binding sites for ProTx-II and HWTX-IV on VSDII overlap, although the interaction details may vary. The L3-4 loop containing residues Ala826-Asp827-Val828-Glu829-Gly830 in VSDII is invisible in both EM reconstructions. Nevertheless, the positions of ProTx-II and HWTX-IV support the previous analysis that the L3-4 loop on VSDII, known as “site 4” (24), is involved in the association of both toxins (3133, 35). Asn774 on S2 may also contribute to the interaction with both toxins (fig. S8, A and B). No direct contact is detected between HWTX-IV and S4II, whereas Leu831 on S4II appears to interact with ProTx-II, which may underlie the further “up” conformation of S4II in the presence of ProTx-II (Fig. 5, B and C, and fig. S8B).

Although the L3-4 loop also represents the major binding site for ProTx-II on VSDIV, the position of the toxin deviates markedly from that in VSDII. Whereas ProTx-II is placed above the central cavity of VSDII and sandwiched by S2II and S3II, the L3-4IV loop (also known as site 3) is solely responsible for binding to ProTx-II, which is oriented away from S2 and makes no contact with other elements in VSDIV (Fig. 5D and fig. S8C).


In both reconstructions, the intracellular gate is closed, and all VSDs exhibit depolarized conformations, although at distinct up states relative to the charge transfer center (48) (fig. S9). Such features are consistent with an inactivated state. In this conformation, the fast-inactivation Ile-Phe-Met motif in the III-IV linker still inserts into the cavity between the S6 helical bundle and the S4-5 restriction ring in repeats III and IV (Fig. 2B), providing further structural support for the “allosteric blocking” mechanism for fast inactivation (17).

The well-characterized ProTx-II and HWTX-IV both effectively inhibit activation of Nav1.7 at ~100 nM (28, 31). They were suggested to bind to VSDII in the resting state (33, 34). Our structures show that they can also bind to the depolarized conformation of VSDII, probably because of the high concentration (50 μM) used for complex reconstitution. ProTx-II was also shown to inhibit inactivation of Nav channels (31); therefore, the binding of depolarized VSDIV is expected. Nonetheless, we are cautious in the interpretation of the observed interactions between the two GMTs and VSDs (fig. S8). It was reported that membranes facilitate the action of these toxins (28, 49, 50). The poor resolution of the toxins may indicate intrinsic flexibility in the absence of lipid bilayer. Structures of Nav channels in complex with these GMTs in a lipid environment, such as nanodisc, will be necessary to provide a more comprehensive understanding of the mode of action of these toxins.

The binding mode for HWTX-IV and ProTx-II by Nav1.7 is distinct from that for Dc1a by NavPaS (27). Whereas HWTX-IV and ProTx-II both bind to the peripheral region that links S3 and S4 in a VSD, Dc1a inserts into the extracellular cavity enclosed by segments in VSDII and the PD (Fig. 5E). Despite this distinction, these GMTs may observe a common principle for function. An entity distinct from the VSDs, such as the PD of NavPaS for Dc1a and the lipid bilayer for HWTX-IV and ProTx-II, provides the support to anchor the GMTs, which then lock the associated VSDs in certain functional states to achieve gating modulation.

Despite many remaining questions, successful recombinant expression, purification, and structural determination of Nav1.7 in the presence of both β1 and β2 subunits and different animal toxins to near-atomic resolution establishes an important framework for investigating the structure-function relationship. Dozens of point mutations that are associated with pain syndromes can now be structurally mapped (table S1) and probed with a precise template. The structures may guide development of therapeutic agents for pain relief.

Supplementary Materials

Materials and Methods

Supplementary Text

Figs. S1 to S9

Tables S1 to S3

References (5368)

References and Notes

Acknowledgments: We thank X. Li (Tsinghua University) for technical support during EM image acquisition. Funding: This work was funded by the National Key Basic Research (973) Program (2015CB910101 to N.Y.) and the National Key R&D Program (2016YFA0500402 to N.Y. and 2016YFA0501100 to J.L.) from the Ministry of Science and Technology of China, and the National Natural Science Foundation of China (projects 31621092, 31630017, and 81861138009 to N.Y.). We thank the Tsinghua University Branch of China National Center for Protein Sciences (Beijing) for providing the cryo-EM facility support. We thank the computational facility support on the cluster of Bio-Computing Platform (Tsinghua University Branch of China National Center for Protein Sciences Beijing) and the “Explorer 100” cluster system of Tsinghua National Laboratory for Information Science and Technology. N.Y. is supported by the Shirley M. Tilghman endowed professorship from Princeton University. Author contributions: N.Y. conceived the project. H.S., D.L., K.W., and J.L. performed the experiments. All authors contributed to data analysis. N.Y. wrote the manuscript. Competing interests: The authors declare no competing interests. Data and materials availability: The EM maps for Nav1.7-HS and Nav1.7-PT have been deposited in EMDB ( with the codes EMD-9781 and EMD-9782, respectively, and the atomic coordinates in the Protein Data Bank ( with the accession codes 6J8G and 6J8H for Nav1.7-HS and 6J8I and 6J8J for Nav1.7-PT.
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