www.sciencemag.org/cgi/content/full/science.aaw2493/DC1

Supplementary Materials for

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

Huaizong Shen*, Dongliang Liu*, Kun Wu, Jianlin Lei, Nieng Yan†

*These authors contributed equally to this work.

†Corresponding author. Email: nyan@princeton.edu

Published 14 February 2019 on Science First Release DOI: 10.1126/science.aaw2493

This PDF file includes:

Materials and Methods Supplementary Text Figs. S1 to S9 Tables S1 to S3 References

Materials and methods

Whole cell electrophysiology

The optimized coding DNAs for Nav1.7 (Uniprot: Q15858) and Nav1.7-E406K were

cloned separately into the pEG BacMam vector with Twin-Strep-tag and FLAG tag in

tandem at the amino terminus while the optimized coding DNAs for β1 (Uniprot: Q07699)

and β2 (Uniprot: O60939) were cloned into the pCAG vector without affinity tag (53, 54).

The HEK293T cells (Invitrogen) were cultured in Dulbecco's Modified Eagle Medium

(DMEM, Gibco) containing 4.5 mg/ml glucose, 10% fetal bovine serum (FBS, Gibco)

and co-transfected with the expression plasmids for Nav1.7 or Nav1.7-E406K with β1 and

β2, and an eGFP-encoding plasmid when cell confluency reached 70%. After incubation

at 37 oC under 5% CO2 for 24 hours, the cells were treated with 0.05% trypsin (Gibco)

and put on poly-D-lysine (Sigma)-coated 10 mm cover slips for whole cell

electrophysiological characterization.

Sodium currents were recorded using the HEKA EPC10 amplifier with Patchmaster

2.90.4 software and glass micropipettes (3-4 MΩ) in HEK293T cells. The data were

analyzed using Fitmaster 2.90.4 (HEKA Elektronik) and Prism 6.02 (GraphPad Software).

For recording the voltage-dependent currents, activation and inactivation currents, the

electrodes were filled with the internal solution composed of (in mM) 40 CsCl, 10 NaCl,

10 EGTA, 105 CsF, 10 HEPES, pH 7.3 with CsOH. The extracellular solution was

composed of (in mM) 130 NaCl, 4 KCl, 1 MgCl2, 1.5 CaCl2, 5 D-Glucose monohydrate,

5 HEPES, pH=7.4 with NaOH.

The voltage dependence of ion current was analyzed using a protocol consisting of steps

from a holding potential of -90 mV to voltages ranging from -100 to 80 mV for 50 ms in

10 mV increment. In the I-V curves, the currents were normalized with the maximum and

plotted against the voltage.

To measure the voltage dependence of activation, the cells were held at -90 mV, and pre-

pulsed at -120 mV for 200 ms. Then, the sodium current was elicited by a stepped

depolarization test voltage pulses from -80 mV to 80 mV for 50 ms with an increment of

5 mV. For the conductance calculation, we used the equation, G= I/(V-Vr), where Vr (the

reversal potential) represents the voltage at which the current is zero. The calculated

conductance was normalized, plotted against the voltage from -80 mV to 0 mV.

2

For the voltage dependence of inactivation, the cells that were clamped at a holding

potential of -90 mV were applied to stepped pre-pulses from -100 mV to 20 mV for 1000

ms with an increment of 5 mV. Then, the sodium current was recorded at the test pulse of

0 mV for 50 ms. The peak currents under the test pulses were normalized and plotted

against the pre-pulse voltage. The extra sum-of-squares F test was used to compare the

V1/2 of activation and inactivation fits. The time course of inactivation data from the peak

current at 0 mV was fitted to a single exponential equation: y = A1 exp(−x/τinac) + y0,

where A1 was the relative fraction of current inactivation, τinac was the time constant, x

was the time, and y0 was the amplitude of the steady-state component. τinac values of

Nav1.7 and Nav1.7 (E406K) were compared by unpaired t-test with Welch’s correction.

Transient co-expression of human Nav1.7 with β1 and β2

Single point mutation E406K was introduced to Nav1.7 using a standard two-step PCR-

based strategy and confirmed by PCR sequencing. The plasmids used for transient

expression of Nav1.7 variants in complex with β1 and β2 are the same as those for

electrophysiological analysis described above. HEK 293F cells (Invitrogen) were

cultured in SMM 293T-I medium (Sino Biological Inc.) under 5% CO2 in a Multitron-Pro

shaker (Infors, 130 r.p.m.) at 37 oC and transfected with plasmids when the cell density

reached 2 × 106 cells per mL. For one litre cell culture, 2.5 mg of plasmids in total (1.5

mg plasmids for Nav1.7 plus 0.5 mg plasmids for β1 and 0.5 mg plasmids for β2) were

pre-incubated with 4 mg 25-kDa linear polyethylenimines (PEIs) (Polysciences) in 25 ml

fresh medium for 15-30 min before transfection, which was initiated by adding the

mixture into cell culture. Transfected cells were cultured for 48 h before harvesting.

Purification of Nav1.7 complexes

16 L transfected cells were harvested by centrifugation at 800 g and resuspended in lysis

buffer containing 25 mM Tris-HCl (pH 7.4) and 150 mM NaCl. The suspension was

supplemented with 1.5% (w/v) n-dodecyl-β-D-maltopyranoside (DDM, Anatrace), 0.3%

(w/v) cholesteryl hemisuccinate Tris salt (CHS, Anatrace), and protease inhibitor cocktail

containing 2 mM phenylmethylsulfonyl fluoride (PMSF), aprotinin (3.9 μg/ml), pepstatin

3

(2.1 μg/ml), and leupeptin (15 μg/ml), and incubated at 4 oC for 2 h. After ultra-

centrifugation at 200,000 g for 40 min, the supernatant was applied to anti-Flag M2

affinity gel (Sigma) by gravity at 4 oC. The resin was rinsed four times with the wash

buffer (buffer W) containing 25 mM Tris-HCl (pH 7.4), 150mM NaCl, 0.04% glyco-

diosgenin (GDN, Anatrace), and protease inhibitor cocktail. The target proteins were

eluted with buffer W plus 200 μg/ml FLAG peptide (Sigma). The eluent was then applied

to Strep-Tactin Sepharose (IBA) and flew through by gravity. The resin was rinsed three

times with buffer W and the target proteins were eluted with buffer W plus 2.5 mM D-

Desthiobiotin (IBA). The eluent was then concentrated using a 100-kDa cut-off Centricon

(Millipore) and further purified by Superose-6 column (GE Healthcare) in buffer W. The

purified proteins were applied to SDS-PAGE and mass spectrometric analysis of the

resolved bands confirmed the presence of all three subunits in the peak fractions. The

purified proteins were pooled and concentrated to approximately 2 mg/ml for cryo-EM

analysis. A combination of ProTx-II (R&D Systems, 50 μM) and TTX (50 μM), or

Huwentoxin-IV (R&D Systems, 50 μM) and STX (11 μM), was separately added to the

concentrated sample half an hour before making cryo grids.

Cryo-EM data acquisition

Aliquots of 3.5 μL freshly purified Nav1.7 complex were placed on glow-discharged 300-

mesh holey carbon grids (Quantifoil, Au, R1.2/1.3), which were blotted for 3.5 s before

being flash-frozen in liquid ethane cooled by liquid nitrogen with Vitrobot Mark IV

(Thermo Fisher). The grids were subsequently transferred to a Titan Krios electron

microscope (Thermo Fisher) operating at 300 kV and equipped with Cs corrector, Gatan

K2 Summit detector, and GIF Quantum energy filter. A total of 5,662 and 4,333 movie

stacks were automatically collected for Nav1.7 complexes supplemented with ProTx-II

and TTX (PT) or Huwentoxin-IV and STX (HS), respectively, using AutoEMation (55)

with a slit width of 20 eV on the energy filter and a preset defocus range from -1.6 µm to

-1.4 µm in super-resolution mode at a nominal magnification of 105,000 X. Each stack

was exposed for 5.6 s with the exposing time of 0.175 s per frame, resulting in a total of

32 frames per stack. The total dose rate was about 48 e-/Å2 for each stack. The stacks

were motion corrected with MotionCor2 (56) and binned 2 fold, resulting in a pixel size

4

of 1.091 Å/pixel. Meanwhile, dose weighting was performed (57). The defocus values

were estimated with Gctf (58).

Image processing

A diagram for the data processing is presented in Figure S3A. A total of 2,207,874 or

1,813,202 particles were automatically picked using RELION (59-61) from 5,662 or

4,333 collected micrographs for Nav1.7-PT or Nav1.7-HS respectively. After 2D

classification, a total of 970,769 or 891,394 good particles for Nav1.7-PT or Na1.7-HS

respectively were selected and subjected to global angular search 3D classification with

K set to 1 (one class). For each of the last 5 iterations, a local angular search 3D

classification was performed, during which the particles were classified into 5 classes. A

total of non-redundant 815,446 or 708,833 particles for Nav1.7-PT or Na1.7-HS

respectively were selected from the local angular searching 3D classification and

subjected to 3 cycles of multi-reference 3D classification to remove bad particles. The

selected particles were subject to 3D auto-refinement, resulting in 3D maps with overall

resolutions of 3.2 Å for both complexes. The final particle numbers used for the 3D auto-

refinement were 263,205 for Nav1.7-PT and 275,630 for Na1.7-HS. 2D classification, 3D

classification, and auto-refinement were performed with RELION 2.1. The resolution

was estimated with the gold-standard Fourier shell correlation 0.143 criterion (62) with

high resolution noise substitution (63).

Model building and structure refinement

Model building was carried out based on the 3.2 Å reconstruction maps. The coordinates

of the human Nav1.4-β1 complex (PDB accession number: 6AGF) were fitted into either

EM map in CHIMERA (64). The sequence of Nav1.4-β1 were mutated to corresponding

residues in Nav1.7-β1 in COOT (65) and every residue was manually checked. The

chemical properties of amino acids were considered during model building. The structure

of β2 (PDB accession number: 5FEB) was docked into the corresponding density in

CHIMERA and the sequence was mutated back to wild type.

In total, 1140 residues for Nav1.7, 173 residues for β1, and 120 residues for β2 were

assigned with side chains, and 10 sugar moieties were built. The N-terminal 113 residues,

5

intracellular I-II linker (residues 418-725), II-III linker (residues 973-1174), and C-

terminal sequences after Glu1768 are invisible in Nav1.7.

Structure refinement was performed using phenix.real_space_refine application in

PHENIX (66) in real space with secondary structure and geometry restraints. Over-fitting

of the overall model was monitored by refining the model in one of the two independent

maps from the gold-standard refinement approach and testing the refined model against

the other map (67). Statistics of the map reconstruction and model refinement can be

found in Table S3. The structures of ProTx-II and Huwentoxin-IV (PDB codes: 5O0U

and 2M4X, respectively) were used for analysis, but cannot be reliably docked.

6

Supplementary Text

Structural shifts between Nav1.7 and Nav1.4

The overall structure of the Nav1.7-1-2 complex is similar to that of the Nav1.4-1

complex (18), including the interface between α and 1 and the conformation of the III-

IV linker, which harbors the fast inactivation motif Ile/Phe/Met (IFM) (68). Structures of

Nav1.7-HS and Nav1.4-1 can be superimposed with a root-mean-square deviation

(RMSD) of 0.7 Å over 1108 Cα atoms (fig. S5A). We will avoid redundant illustration of

the identical features, but focus on the structural distinctions.

Minor structural shifts are observed at the extracellular loops of repeats I and III,

the extracellular tips of VSDII, and the intracellular segments of VSDIV between Nav1.7

and Nav1.4 (fig. S5A).The extracellular loop of repeat I is longer in Nav1.4 than in other

Navs. The segment 287-335 is invisible in the structure of Nav1.4 (fig. S1). But the

corresponding shorter sequence in Nav1.7 (residues 281-302) is completely resolved and

folds to a helical hairpin (fig. S5B, left). The extracellular region in repeat III undergoes

an overall drift, while that in repeats II and IV can be completely superimposed between

the two channel structures (fig. S5B, right).

As VSDII provides the docking site for ProTx-II and HWTX-IV, the structural

shifts of this domain between Nav1.7 and Nav1.4 might be explained by the docking of

the toxins. Five residues that connect the S3 and S4 segments (the L3-4II loop) are

invisible in Nav1.7. Both extracellular tips of S3 and S4 slightly bend away from S1 and

S2 in Nav1.7 relative to those in Nav1.4 (fig. S5C). A major conformational shift occurs

to the gating charge (GC) residue R2 (Arg835 in Nav1.7 and Arg669 in Nav1.4), the

helical turn carrying which is an α helix in Nav1.4 and a 310 helix in Nav1.7 (fig. S5C,

right).

7

Supplementary Figures and Legends

8

9

Fig. S1 Sequence alignment of the nine subtypes of human Nav channels.

The figure is adapted from our recent publication (18) with minor adjustment. The secondary elements of human Nav1.7 are shown above the alignment. The secondary structure for the C-terminal domain is predicted from the structure of NavPaS (16). Invariant amino acids are shaded yellow and conserved residues are colored blue. The critical DEKA residues are colored red and shaded yellow. The gating charge residues (labeled R/K1-6) in the S4 segment of each repeat are colored white and shaded orange. The residues on S1-S3 in each repeat that may facilitate gating charge transfer are shaded cyan. Cys895 in Nav1.7, which is responsible for disulfide-bond formation with Cys55 in β2 is highlighted. The brown square indicates Glu406 that was mutated to Lys in this study. The Uniprot IDs for the aligned sequences are: hNav1.1: P35498; hNav1.2: Q99250; hNav1.3: Q9NY46; hNav1.4: P35499; hNav1.5: Q14524; hNav1.6: Q9UQD0; hNav1.7: Q15858; hNav1.8: Q9Y5Y9; hNav1.9: Q9UI33.

10

Fig. S2 Electrophysiological properties of Nav1.7 transiently co-expressed with the β1and β2 subunits in HEK293T cells.

(A) Voltage-dependent ion current traces. (B) Voltage-dependent activation traces. (C) Voltage-dependent inactivation traces. The left panels show the diagrams of recording protocols. The traces for wild type Nav1.7 and Nav1.7 (E406K) in the presence of β1and β2 are shown in the middle and right panels, respectively. Please refer to Methods for details.

11

Fig. S3 Cryo-EM structural analysis of the human Nav1.7-β1-β2 complex in the presence of ProTx-2/TTX (Nav1.7-PT) or HWTX-IV/STX (Nav1.7-HS).

(A) The flowchart for EM data processing. Details can be found in Materials and Methods. (B) FSC curves of the refined model versus the overall map that it was refined against (black), of the model refined in the first of the two independent maps used for the gold-standard FSC versus that same map (red), and of the model refined in the first of the two independent maps versus the second independent map (green). The small difference between the red and green curves indicates that the refinement of the atomic coordinates did not suffer from overfitting.

12

Fig. S4 EM maps for representative segments of the Nav1.7 complexes.

(A) Local resolution maps of Nav1.7-PT (left) and Nav1.7-HS (right). (B) EM maps for the S1-S6 segments in each repeat of Nav1.7-HS. (C) EM maps for the S3 and S4 segments in VSDII of Nav1.7-PT. The rest of the map is nearly identical between Nav1.7-PT and Nav1.7-HS, therefore not shown for Nav1.7-PT. The maps were prepared in PyMol and contoured at 3 .

13

Fig. S5

Structural shifts between Nav1.7 and Nav1.4.

(A) Structures of Nav1.7-HS and Nav1.4-β1 can be superimposed with a RMSD of 0.716 Å overall 1108 Cα atoms. The structural deviations are indicated by pink shadows. Nav1.7-HS is color-coded and Nav1.4-β1 (PDB code: 6AGF) is colored pale pink. (B) Structural differences in the extracellular loops between Nav1.7 and Nav1.4. Whereas the extracellular loops in repeats II and repeats IV are identical in the two structures, those in repeats I and III exhibit some deviations. In particular, Nav1.7 has two short helices (designated Eα1a and Eα1b) in the extracellular region of repeats I, Nav1.4 has only one preceding a Nav1.4-unique segment that was unresolved in the EM map. Red arrows indicate the conformational shifts of the corresponding segments from Nav1.4 to Nav1.7. (C) Major structural differences in VSDII. The S4 segment in VSDII exhibits distinct “up” states in Nav1.7-HS (pale green), Nav1.7-PT (white), and Nav1.4 (pale pink). While the helical turn carrying R2 is relaxed to an α helical turn in Nav1.4, the entire S4 segment in both Nav1.7 structures remains to be 310 helix (right panel).

14

Fig. S6 Side chain rotation of Phe387 and Phe391 on S6I leads to closure of one fenestration.

(A) Met1754 and Tyr1755 on S6IV exist as two rotamers in the 3D reconstructions. (B) The density for Phe387 and Phe391 on S6I. The local densities for the indicated residues, shown as blue mesh, are contoured at 3 (C) Rotation of the aromatic rings of Phe387 and Phe391 seals the fenestration on the side wall constituted by repeats I and IV. Left: Surface electrostatic potential of Nav1.7-HS calculated in PyMol. Right: Distinct side chain conformations of Phe387 and Phe391 in Nav1.7 (grey) from the corresponding residues in Nav1.4 (pale pink, thin sticks).

15

Fig. S7 EM maps for TTX/STX and the surrounding elements.

(A) Stereoviews of the densities for STX and TTX contoured at 5 . (B,C) The EM maps for the P1-SF-P2 segment in each repeat in Nav1.7-HS (B) and Nav1.7-PT (C). The densities for STX and TTX are shown in each panel to indicate the relative position of these blockers to the SF. The SF signature motif residues DEKA are highlighted by colored shadows. The densities were prepared in PyMol and contoured at 3 in panels B and C.

16

Fig. S8 Binding sites for HWTX-IV and ProTx-II.

(A, B) HWTX-IV and ProTx-II bind similarly to site 4 on Nav1.7-VSDII. Two views are shown in the left and middle panels, and the residues on S2-S4 segments that may be involved in toxin binding are shown as sticks in the right panels. In both EM reconstructions, residues 826ADVEG830 that constitute site 4 (the L3-4 loop of VSDII) are invisible. (C) Binding site for ProTx-II on VSDIV. Residues on the L3-4 loop (also defined as site 3) that may participate in ProTx-II binding are shown as sticks.

17

Fig. S9 All four VSDs exhibit “up” conformations.

(A) Structures of the four VSDs in Nav1.7-HS. The S1 segment in each VSD is omitted for visual clarity. It is noted that the helical turn that carries R1 in VSDIV exists as an α helix, while all other GC residues in the four repeats are located on 310 helices. (B) The GC residues exhibit distinct “up” conformations relative to the charge transfer center (CTC) in the four repeats (48). The four VSDs are superimposed relative to CTC. For visual clarity, S1 and S2 are omitted. The Cα atoms of GCs are shown as spheres in the lower panel. The positions of the Cα atoms of the indicated GC residues in VSDs I-IV are indicated by black, green, orange, and cyan lines, respectively. Both the Cα atoms and even more the side chains of GC residues stay in different heights relative to the CTC.

18

Table S1 Structural mapping of disease-related mutations identified in human Nav1.7

Mutations Disease Structure Mutations Disease StructureQ10R PERYTHM NTD V1309F PEPD S4-5III I136V PERYTHM S1I V1310F PEPD S4-5III S211P PERYTHM S4I I1472T PEPD III-IV F216S PERYTHM S4I F1473V PEPD III-IV I234T PERYTHM S4-5I T1475I PEPD III-IV S241T PERYTHM S4-5I G1618R PEPD S4IV L245V PERYTHM S5I L1623P PEPD S4IV N395K PERYTHM S6I M1638K PEPD S4-5IV V400M PERYTHM S6I A1643E PEPD S5IV E406K PERYTHM S6I N641Y GEFS+7 I-II linker P610T PERYTHM I-II linker K666R GEFS+7 I-II linker G616R PERYTHM I-II linker I62V febrile seizures NTD L834R PERYTHM S4II P149Q febrile seizures S1-2I I859T PERYTHM S4-5II S490N febrile seizures I-II linker G867D PERYTHM S5II N641Y febrile seizures I-II linker L869F PERYTHM S5II K666R febrile seizures I-II linker L869H PERYTHM S5II I750V febrile seizures S1II A874P PERYTHM S5II I228M Dravet syndrome S4IV883G PERYTHM S5II E519K Dravet syndrome I-II linker Q886E PERYTHM S5II K666R Dravet syndrome I-II linker DL966 PERYTHM S6II I695M Dravet syndrome I-II linker P1319L PERYTHM S5III C710Y Dravet syndrome I-II linker F1460V PERYTHM S6III I750V Dravet syndrome S1IIA1643E PERYTHM S5IV L1134F Dravet syndrome II-III linkerA1643G PERYTHM S5IV E1171Q Dravet syndrome II-III linkerA1643T PERYTHM S5IV L1278V Dravet syndrome S3IIIR907Q CIP extracellular II I228M SFSN S4I A1247E CIP S3III D623N SFSN I-II linker

D1381-1385 CIP Eb3c III I731K SFSN S0II W1786R CIP CTD I750V SFSN S1II R185H PEPD S2-3I M943L/V1002L SFSN S6II / II-IIIR1007C PEPD II-III linker M1543I SFSN S2IV V1309D PEPD S4-5III W1550R CNP S2IV

PERYTHM: Primary erythermalgia; CIP: Congenital indifference to pain; PEPD: Paroxysmal extreme pain disorder; GEFS+7: Generalized epilepsy with febrile seizures plus 7; SFSN: Idiopathic small fiber sensory neuropathy; CNP: chronic neuropathic pain. The table is adapted from (36).

19

Table S2 Activation and steady-state inactivation parameters of Nav1.7 variants transiently co-expressed with β1 and β1 in HEK293T cells

* P < 0.05 versus WT, *** P < 0.001 versus WT, **** P < 0.0001 versus WT. Each datapoint represents mean ± s.e.m (standard deviation of mean). The extra sum-of-squares F test was used to compare the V1/2 of activation and inactivation fits. τinac values of Nav1.7 and Nav1.7-E406K were compared by unpaired t-test with Welch’s correction.

Activation Curve Steady-State Inactivation Curve V1/2 k n V1/2 k Τinac (ms) n

Nav1.7 -25.19±0.47 5.55±0.41 5 -78.66±0.72 6.41±0.57 0.80±0.08 5

Nav1.7-E406K

-35.81±0.63**** 5.63±0.57 11 -73.69±0.88*** 8.45±0.70 2.07±0.45* 12

20

Table S3 Statistics for data collection and structural refinement.

Data collection Nav1.7-PT Nav1.7-HS EM equipment Titan Krios (Thermo Fisher Scientific Inc.) Voltage (kV) 300 Detector Gatan K2 Summit Energy filter Gatan GIF Quantum, 20 eV slit Pixel size (Å) 1.091 Electron dose (e-/Å2) 48

Defocus range (μm) -1.0 ~ -2.5 Number of collected movie stacks

5,662 4,333

Reconstruction Software RELION 2.1 Number of used particles 263,205 275,630

Symmetry C1 Overall resolution (Å) 3.2 3.2 Map sharpening B-factor (Å2) -120 -120

Refinement Software Phenix Cell dimensions

a=b=c (Å) α== (˚)

261.84 90

261.84 90

Model composition Protein residues 1433 1433 Side chains assigned 1433 1433 Sugar 11 11

R.m.s deviations Bonds length (Å) 0.01 0.01 Bonds angle (˚) 1.04 1.02

Ramachandran plot statistics (%)

Preferred 95.2 94.7Allowed 4.50 5.1Outlier 0.3 0.2

21

References and Notes

1. N. Klugbauer, L. Lacinova, V. Flockerzi, F. Hofmann, Structure and functional expression of a new member of the tetrodotoxin-sensitive voltage-activated sodium channel family from human neuroendocrine cells. EMBO J. 14, 1084–1090 (1995). doi:10.1002/j.1460-2075.1995.tb07091.x Medline

2. S. D. Dib-Hajj, T. R. Cummins, J. A. Black, S. G. Waxman, From genes to pain: Nav1.7 and human pain disorders. Trends Neurosci. 30, 555–563 (2007). doi:10.1016/j.tins.2007.08.004 Medline

3. A. I. Basbaum, D. M. Bautista, G. Scherrer, D. Julius, Cellular and molecular mechanisms of pain. Cell 139, 267–284 (2009). doi:10.1016/j.cell.2009.09.028 Medline

4. D. L. Bennett, C. G. Woods, Painful and painless channelopathies. Lancet Neurol. 13, 587–599 (2014). doi:10.1016/S1474-4422(14)70024-9 Medline

5. M. A. Nassar, L. C. Stirling, G. Forlani, M. D. Baker, E. A. Matthews, A. H. Dickenson, J. N. Wood, Nociceptor-specific gene deletion reveals a major role for Nav1.7 (PN1) in acute and inflammatory pain. Proc. Natl. Acad. Sci. U.S.A. 101, 12706–12711 (2004). doi:10.1073/pnas.0404915101 Medline

6. Y. Yang, Y. Wang, S. Li, Z. Xu, H. Li, L. Ma, J. Fan, D. Bu, B. Liu, Z. Fan, G. Wu, J. Jin, B. Ding, X. Zhu, Y. Shen, Mutations in SCN9A, encoding a sodium channel alpha subunit, in patients with primary erythermalgia. J. Med. Genet. 41, 171–174 (2004). doi:10.1136/jmg.2003.012153 Medline

7. T. R. Cummins, S. D. Dib-Hajj, S. G. Waxman, Electrophysiological properties of mutant Nav1.7 sodium channels in a painful inherited neuropathy. J. Neurosci. 24, 8232–8236 (2004). doi:10.1523/JNEUROSCI.2695-04.2004 Medline

8. J. J. Cox, F. Reimann, A. K. Nicholas, G. Thornton, E. Roberts, K. Springell, G. Karbani, H. Jafri, J. Mannan, Y. Raashid, L. Al-Gazali, H. Hamamy, E. M. Valente, S. Gorman, R. Williams, D. P. McHale, J. N. Wood, F. M. Gribble, C. G. Woods, An SCN9A channelopathy causes congenital inability to experience pain. Nature 444, 894–898 (2006). doi:10.1038/nature05413 Medline

9. C. R. Fertleman, M. D. Baker, K. A. Parker, S. Moffatt, F. V. Elmslie, B. Abrahamsen, J. Ostman, N. Klugbauer, J. N. Wood, R. M. Gardiner, M. Rees, SCN9A mutations in paroxysmal extreme pain disorder: Allelic variants underlie distinct channel defects and phenotypes. Neuron 52, 767–774 (2006). doi:10.1016/j.neuron.2006.10.006 Medline

10. Y. P. Goldberg, J. MacFarlane, M. L. MacDonald, J. Thompson, M.-P. Dube, M. Mattice, R. Fraser, C. Young, S. Hossain, T. Pape, B. Payne, C. Radomski, G. Donaldson, E. Ives, J. Cox, H. B. Younghusband, R. Green, A. Duff, E. Boltshauser, G. A. Grinspan, J. H. Dimon, B. G. Sibley, G. Andria, E. Toscano, J. Kerdraon, D. Bowsher, S. N. Pimstone, M. E. Samuels, R. Sherrington, M. R. Hayden, Loss-of-function mutations in the Nav1.7 gene underlie congenital

22

indifference to pain in multiple human populations. Clin. Genet. 71, 311–319 (2007). doi:10.1111/j.1399-0004.2007.00790.x Medline

11. E. C. Emery, A. P. Luiz, J. N. Wood, Nav1.7 and other voltage-gated sodium channels as drug targets for pain relief. Expert Opin. Ther. Targets 20, 975–983 (2016). doi:10.1517/14728222.2016.1162295 Medline

12. Y. P. Goldberg, N. Price, R. Namdari, C. J. Cohen, M. H. Lamers, C. Winters, J. Price, C. E. Young, H. Verschoof, R. Sherrington, S. N. Pimstone, M. R. Hayden, Treatment of Nav1.7-mediated pain in inherited erythromelalgia using a novel sodium channel blocker. Pain 153, 80–85 (2012). doi:10.1016/j.pain.2011.09.008 Medline

13. J. H. Lee, C.-K. Park, G. Chen, Q. Han, R.-G. Xie, T. Liu, R.-R. Ji, S.-Y. Lee, A monoclonal antibody that targets a Nav1.7 channel voltage sensor for pain and itch relief. Cell 157, 1393–1404 (2014). doi:10.1016/j.cell.2014.03.064 Medline

14. W. A. Catterall, A. L. Goldin, S. G. Waxman, International Union of Pharmacology. XLVII. Nomenclature and structure-function relationships of voltage-gated sodium channels. Pharmacol. Rev. 57, 397–409 (2005). doi:10.1124/pr.57.4.4 Medline

15. M. Noda, S. Shimizu, T. Tanabe, T. Takai, T. Kayano, T. Ikeda, H. Takahashi, H. Nakayama, Y. Kanaoka, N. Minamino, K. Kangawa, H. Matsuo, M. A. Raftery, T. Hirose, S. Inayama, H. Hayashida, T. Miyata, S. Numa, Primary structure of Electrophorus electricus sodium channel deduced from cDNA sequence. Nature 312, 121–127 (1984). doi:10.1038/312121a0 Medline

16. H. Shen, Q. Zhou, X. Pan, Z. Li, J. Wu, N. Yan, Structure of a eukaryotic voltage-gated sodium channel at near-atomic resolution. Science 355, eaal4326 (2017). doi:10.1126/science.aal4326 Medline

17. Z. Yan, Q. Zhou, L. Wang, J. Wu, Y. Zhao, G. Huang, W. Peng, H. Shen, J. Lei, N. Yan, Structure of the Nav1.4-β1 complex from electric eel. Cell 170, 470–482.e11 (2017). doi:10.1016/j.cell.2017.06.039 Medline

18. X. Pan, Z. Li, Q. Zhou, H. Shen, K. Wu, X. Huang, J. Chen, J. Zhang, X. Zhu, J. Lei, W. Xiong, H. Gong, B. Xiao, N. Yan, Structure of the human voltage-gated sodium channel Nav1.4 in complex with β1. Science 362, eaau2486 (2018). doi:10.1126/science.aau2486 Medline

19. S. B. Long, E. B. Campbell, R. Mackinnon, Crystal structure of a mammalian voltage-dependent Shaker family K+ channel. Science 309, 897–903 (2005). doi:10.1126/science.1116269 Medline

20. F. H. Yu, V. Yarov-Yarovoy, G. A. Gutman, W. A. Catterall, Overview of molecular relationships in the voltage-gated ion channel superfamily. Pharmacol. Rev. 57, 387–395 (2005). doi:10.1124/pr.57.4.13 Medline

21. H. Terlau, S. H. Heinemann, W. Stühmer, M. Pusch, F. Conti, K. Imoto, S. Numa, Mapping the site of block by tetrodotoxin and saxitoxin of sodium channel II. FEBS Lett. 293, 93–96 (1991). doi:10.1016/0014-5793(91)81159-6 Medline

23

22. H. A. O’Malley, L. L. Isom, Sodium channel β subunits: Emerging targets in channelopathies. Annu. Rev. Physiol. 77, 481–504 (2015). doi:10.1146/annurev-physiol-021014-071846 Medline

23. C. J. Laedermann, N. Syam, M. Pertin, I. Decosterd, H. Abriel, β1- and β3- voltage-gated sodium channel subunits modulate cell surface expression and glycosylation of Nav1.7 in HEK293 cells. Front. Cell. Neurosci. 7, 137 (2013). doi:10.3389/fncel.2013.00137 Medline

24. W. A. Catterall, S. Cestèle, V. Yarov-Yarovoy, F. H. Yu, K. Konoki, T. Scheuer, Voltage-gated ion channels and gating modifier toxins. Toxicon 49, 124–141 (2007). doi:10.1016/j.toxicon.2006.09.022 Medline

25. F. Zhang, X. Xu, T. Li, Z. Liu, Shellfish toxins targeting voltage-gated sodium channels. Mar. Drugs 11, 4698–4723 (2013). doi:10.3390/md11124698 Medline

26. S. Yang, Y. Xiao, D. Kang, J. Liu, Y. Li, E. A. B. Undheim, J. K. Klint, M. Rong, R. Lai, G. F. King, Discovery of a selective Nav1.7 inhibitor from centipede venom with analgesic efficacy exceeding morphine in rodent pain models. Proc. Natl. Acad. Sci. U.S.A. 110, 17534–17539 (2013). doi:10.1073/pnas.1306285110 Medline

27. H. Shen, Z. Li, Y. Jiang, X. Pan, J. Wu, B. Cristofori-Armstrong, J. J. Smith, Y. K. Y. Chin, J. Lei, Q. Zhou, G. F. King, N. Yan, Structural basis for the modulation of voltage-gated sodium channels by animal toxins. Science 362, eaau2596 (2018). doi:10.1126/science.aau2596 Medline

28. K. Peng, Q. Shu, Z. Liu, S. Liang, Function and solution structure of huwentoxin-IV, a potent neuronal tetrodotoxin (TTX)-sensitive sodium channel antagonist from Chinese bird spider Selenocosmia huwena. J. Biol. Chem. 277, 47564–47571 (2002). doi:10.1074/jbc.M204063200 Medline

29. R. E. Middleton, V. A. Warren, R. L. Kraus, J. C. Hwang, C. J. Liu, G. Dai, R. M. Brochu, M. G. Kohler, Y.-D. Gao, V. M. Garsky, M. J. Bogusky, J. T. Mehl, C. J. Cohen, M. M. Smith, Two tarantula peptides inhibit activation of multiple sodium channels. Biochemistry 41, 14734–14747 (2002). doi:10.1021/bi026546a Medline

30. W. A. Schmalhofer, J. Calhoun, R. Burrows, T. Bailey, M. G. Kohler, A. B. Weinglass, G. J. Kaczorowski, M. L. Garcia, M. Koltzenburg, B. T. Priest, ProTx-II, a selective inhibitor of Nav1.7 sodium channels, blocks action potential propagation in nociceptors. Mol. Pharmacol. 74, 1476–1484 (2008). doi:10.1124/mol.108.047670 Medline

31. Y. Xiao, K. Blumenthal, J. O. Jackson 2nd, S. Liang, T. R. Cummins, The tarantula toxins ProTx-II and huwentoxin-IV differentially interact with human Nav1.7 voltage sensors to inhibit channel activation and inactivation. Mol. Pharmacol. 78, 1124–1134 (2010). doi:10.1124/mol.110.066332 Medline

32. Y. Xiao, J. O. Jackson 2nd, S. Liang, T. R. Cummins, Common molecular determinants of tarantula huwentoxin-IV inhibition of Na+ channel voltage sensors in domains II and IV. J. Biol. Chem. 286, 27301–27310 (2011). doi:10.1074/jbc.M111.246876 Medline

24

33. Y. Xiao, J.-P. Bingham, W. Zhu, E. Moczydlowski, S. Liang, T. R. Cummins, Tarantula huwentoxin-IV inhibits neuronal sodium channels by binding to receptor site 4 and trapping the domain II voltage sensor in the closed configuration. J. Biol. Chem. 283, 27300–27313 (2008). doi:10.1074/jbc.M708447200 Medline

34. S. Sokolov, R. L. Kraus, T. Scheuer, W. A. Catterall, Inhibition of sodium channel gating by trapping the domain II voltage sensor with protoxin II. Mol. Pharmacol. 73, 1020–1028 (2008). doi:10.1124/mol.107.041046 Medline

35. F. Bosmans, M. F. Martin-Eauclaire, K. J. Swartz, Deconstructing voltage sensor function and pharmacology in sodium channels. Nature 456, 202–208 (2008). doi:10.1038/nature07473 Medline

36. W. Huang, M. Liu, S. F. Yan, N. Yan, Structure-based assessment of disease-related mutations in human voltage-gated sodium channels. Protein Cell 8, 401–438 (2017). doi:10.1007/s13238-017-0372-z Medline

37. S. D. Dib-Hajj, A. M. Rush, T. R. Cummins, F. M. Hisama, S. Novella, L. Tyrrell, L. Marshall, S. G. Waxman, Gain-of-function mutation in Nav1.7 in familial erythromelalgia induces bursting of sensory neurons. Brain 128, 1847–1854 (2005). doi:10.1093/brain/awh514 Medline

38. C. Han, S. D. Dib-Hajj, Z. Lin, Y. Li, E. M. Eastman, L. Tyrrell, X. Cao, Y. Yang, S. G. Waxman, Early- and late-onset inherited erythromelalgia: Genotype-phenotype correlation. Brain 132, 1711–1722 (2009). doi:10.1093/brain/awp078 Medline

39. M. Eberhardt, J. Nakajima, A. B. Klinger, C. Neacsu, K. Hühne, A. O. O’Reilly, A. M. Kist, A. K. Lampe, K. Fischer, J. Gibson, C. Nau, A. Winterpacht, A. Lampert, Inherited pain: Sodium channel Nav1.7 A1632T mutation causes erythromelalgia due to a shift of fast inactivation. J. Biol. Chem. 289, 1971–1980 (2014). doi:10.1074/jbc.M113.502211 Medline

40. J. D. Kapplinger, D. J. Tester, B. A. Salisbury, J. L. Carr, C. Harris-Kerr, G. D. Pollevick, A. A. M. Wilde, M. J. Ackerman, Spectrum and prevalence of mutations from the first 2,500 consecutive unrelated patients referred for the FAMILION long QT syndrome genetic test. Heart Rhythm 6, 1297–1303 (2009). doi:10.1016/j.hrthm.2009.05.021 Medline

41. J. D. Kapplinger, D. J. Tester, M. Alders, B. Benito, M. Berthet, J. Brugada, P. Brugada, V. Fressart, A. Guerchicoff, C. Harris-Kerr, S. Kamakura, F. Kyndt, T. T. Koopmann, Y. Miyamoto, R. Pfeiffer, G. D. Pollevick, V. Probst, S. Zumhagen, M. Vatta, J. A. Towbin, W. Shimizu, E. Schulze-Bahr, C. Antzelevitch, B. A. Salisbury, P. Guicheney, A. A. M. Wilde, R. Brugada, J.-J. Schott, M. J. Ackerman, An international compendium of mutations in the SCN5A-encoded cardiac sodium channel in patients referred for Brugada syndrome genetic testing. Heart Rhythm 7, 33–46 (2010). doi:10.1016/j.hrthm.2009.09.069 Medline

42. C. Napolitano, S. G. Priori, P. J. Schwartz, R. Bloise, E. Ronchetti, J. Nastoli, G. Bottelli, M. Cerrone, S. Leonardi, Genetic testing in the long QT syndrome: Development and validation of an efficient approach to genotyping in clinical practice. JAMA 294, 2975–2980 (2005). doi:10.1001/jama.294.23.2975 Medline

25

43. G. Fukuma, H. Oguni, Y. Shirasaka, K. Watanabe, T. Miyajima, S. Yasumoto, M. Ohfu, T. Inoue, A. Watanachai, R. Kira, M. Matsuo, H. Muranaka, F. Sofue, B. Zhang, S. Kaneko, A. Mitsudome, S. Hirose, Mutations of neuronal voltage-gated Na+ channel α1 subunit gene SCN1A in core severe myoclonic epilepsy in infancy (SMEI) and in borderline SMEI (SMEB). Epilepsia 45, 140–148 (2004). doi:10.1111/j.0013-9580.2004.15103.x Medline

44. N. A. Hagen, P. du Souich, B. Lapointe, M. Ong-Lam, B. Dubuc, D. Walde, R. Love, A. H. Ngoc; Canadian Tetrodotoxin Study Group, Tetrodotoxin for moderate to severe cancer pain: A randomized, double blind, parallel design multicenter study. J. Pain Symptom Manage. 35, 420–429 (2008). doi:10.1016/j.jpainsymman.2007.05.011 Medline

45. F. R. Nieto, E. J. Cobos, M. Á. Tejada, C. Sánchez-Fernández, R. González-Cano, C. M. Cendán, Tetrodotoxin (TTX) as a therapeutic agent for pain. Mar. Drugs 10, 281–305 (2012). doi:10.3390/md10020281 Medline

46. M. Chorny, R. J. Levy, Site-specific analgesia with sustained release liposomes. Proc. Natl. Acad. Sci. U.S.A. 106, 6891–6892 (2009). doi:10.1073/pnas.0903079106 Medline

47. J. R. Walker, P. A. Novick, W. H. Parsons, M. McGregor, J. Zablocki, V. S. Pande, J. Du Bois, Marked difference in saxitoxin and tetrodotoxin affinity for the human nociceptive voltage-gated sodium channel (Nav1.7) [corrected]. Proc. Natl. Acad. Sci. U.S.A. 109, 18102–18107 (2012). doi:10.1073/pnas.1206952109 Medline

48. X. Tao, A. Lee, W. Limapichat, D. A. Dougherty, R. MacKinnon, A gating charge transfer center in voltage sensors. Science 328, 67–73 (2010). doi:10.1126/science.1185954 Medline

49. S. T. Henriques, E. Deplazes, N. Lawrence, O. Cheneval, S. Chaousis, M. Inserra, P. Thongyoo, G. F. King, A. E. Mark, I. Vetter, D. J. Craik, C. I. Schroeder, Interaction of tarantula venom peptide ProTx-II with lipid membranes is a prerequisite for its inhibition of human voltage-gated sodium channel Nav1.7. J. Biol. Chem. 291, 17049–17065 (2016). doi:10.1074/jbc.M116.729095 Medline

50. A. J. Agwa, N. Lawrence, E. Deplazes, O. Cheneval, R. M. Chen, D. J. Craik, C. I. Schroeder, S. T. Henriques, Spider peptide toxin HwTx-IV engineered to bind to lipid membranes has an increased inhibitory potency at human voltage-gated sodium channel hNaV1.7. Biochimica et biophysica Acta Biomembranes 1859, 835–844 (2017). doi:10.1016/j.bbamem.2017.01.020 Medline

51. W. L. DeLano, The PyMOL Molecular Graphics System (Schrödinger, 2002); www.pymol.org.

52. O. S. Smart, J. G. Neduvelil, X. Wang, B. A. Wallace, M. S. Sansom, HOLE: A program for the analysis of the pore dimensions of ion channel structural models. J. Mol. Graph. 14, 354–360, 376 (1996). doi:10.1016/S0263-7855(97)00009-X Medline

26

53. A. Goehring, C.-H. Lee, K. H. Wang, J. C. Michel, D. P. Claxton, I. Baconguis, T. Althoff, S. Fischer, K. C. Garcia, E. Gouaux, Screening and large-scale expression of membrane proteins in mammalian cells for structural studies. Nat. Protoc. 9, 2574–2585 (2014). doi:10.1038/nprot.2014.173 Medline

54. T. Matsuda, C. L. Cepko, Electroporation and RNA interference in the rodent retina in vivo and in vitro. Proc. Natl. Acad. Sci. U.S.A. 101, 16–22 (2004). doi:10.1073/pnas.2235688100 Medline

55. J. Lei, J. Frank, Automated acquisition of cryo-electron micrographs for single particle reconstruction on an FEI Tecnai electron microscope. J. Struct. Biol. 150, 69–80 (2005). doi:10.1016/j.jsb.2005.01.002 Medline

56. S. Q. Zheng, E. Palovcak, J.-P. Armache, K. A. Verba, Y. Cheng, D. A. Agard, MotionCor2: Anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat. Methods 14, 331–332 (2017). doi:10.1038/nmeth.4193 Medline

57. T. Grant, N. Grigorieff, Measuring the optimal exposure for single particle cryo-EM using a 2.6 Å reconstruction of rotavirus VP6. eLife 4, e06980 (2015). doi:10.7554/eLife.06980 Medline

58. K. Zhang, Gctf: Real-time CTF determination and correction. J. Struct. Biol. 193, 1–12 (2016). doi:10.1016/j.jsb.2015.11.003 Medline

59. S. H. Scheres, RELION: Implementation of a Bayesian approach to cryo-EM structure determination. J. Struct. Biol. 180, 519–530 (2012). doi:10.1016/j.jsb.2012.09.006 Medline

60. S. H. Scheres, Semi-automated selection of cryo-EM particles in RELION-1.3. J. Struct. Biol. 189, 114–122 (2015). doi:10.1016/j.jsb.2014.11.010 Medline

61. D. Kimanius, B. O. Forsberg, S. H. Scheres, E. Lindahl, Accelerated cryo-EM structure determination with parallelisation using GPUs in RELION-2. eLife 5, e18722 (2016). doi:10.7554/eLife.18722 Medline

62. P. B. Rosenthal, R. Henderson, Optimal determination of particle orientation, absolute hand, and contrast loss in single-particle electron cryomicroscopy. J. Mol. Biol. 333, 721–745 (2003). doi:10.1016/j.jmb.2003.07.013 Medline

63. S. Chen, G. McMullan, A. R. Faruqi, G. N. Murshudov, J. M. Short, S. H. W. Scheres, R. Henderson, High-resolution noise substitution to measure overfitting and validate resolution in 3D structure determination by single particle electron cryomicroscopy. Ultramicroscopy 135, 24–35 (2013). doi:10.1016/j.ultramic.2013.06.004 Medline

64. E. F. Pettersen, T. D. Goddard, C. C. Huang, G. S. Couch, D. M. Greenblatt, E. C. Meng, T. E. Ferrin, UCSF Chimera—A visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004). doi:10.1002/jcc.20084 Medline

27

65. P. Emsley, B. Lohkamp, W. G. Scott, K. Cowtan, Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010). doi:10.1107/S0907444910007493 Medline

66. P. D. Adams, P. V. Afonine, G. Bunkóczi, V. B. Chen, I. W. Davis, N. Echols, J. J. Headd, L. W. Hung, G. J. Kapral, R. W. Grosse-Kunstleve, A. J. McCoy, N. W. Moriarty, R. Oeffner, R. J. Read, D. C. Richardson, J. S. Richardson, T. C. Terwilliger, P. H. Zwart, PHENIX: A comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010). doi:10.1107/S0907444909052925 Medline

67. A. Amunts, A. Brown, X. C. Bai, J. L. Llácer, T. Hussain, P. Emsley, F. Long, G. Murshudov, S. H. W. Scheres, V. Ramakrishnan, Structure of the yeast mitochondrial large ribosomal subunit. Science 343, 1485–1489 (2014). doi:10.1126/science.1249410 Medline

68. J. W. West, D. E. Patton, T. Scheuer, Y. Wang, A. L. Goldin, W. A. Catterall, A cluster of hydrophobic amino acid residues required for fast Na+-channel inactivation. Proc. Natl. Acad. Sci. U.S.A. 89, 10910–10914 (1992). doi:10.1073/pnas.89.22.10910 Medline

28