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Structural basis for NaV1.7 inhibition by pore blockers

Abstract

Voltage-gated sodium channel NaV1.7 plays essential roles in pain and odor perception. NaV1.7 variants cause pain disorders. Accordingly, NaV1.7 has elicited extensive attention in developing new analgesics. Here we present cryo-EM structures of human NaV1.7/β1/β2 complexed with inhibitors XEN907, TC-N1752 and NaV1.7-IN2, explaining specific binding sites and modulation mechanism for the pore blockers. These inhibitors bind in the central cavity blocking ion permeation, but engage different parts of the cavity wall. XEN907 directly causes α- to π-helix transition of DIV-S6 helix, which tightens the fast inactivation gate. TC-N1752 induces π-helix transition of DII-S6 helix mediated by a conserved asparagine on DIII-S6, which closes the activation gate. NaV1.7-IN2 serves as a pore blocker without causing conformational change. Electrophysiological results demonstrate that XEN907 and TC-N1752 stabilize NaV1.7 in inactivated state and delay the recovery from inactivation. Our results provide structural framework for NaV1.7 modulation by pore blockers, and important implications for developing subtype-selective analgesics.

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Fig. 1: The NTD structure of NaV1.7.
Fig. 2: Structural basis for NaV1.7 modulation by XEN907.
Fig. 3: Structural basis for NaV1.7 modulation by TC-N1752.
Fig. 4: Structural basis for pore blocking of NaV1.7 by NaV1.7-IN2.
Fig. 5: Drug-binding sites in the central cavity of NaV channels.

Data availability

The UniProt accession codes for the sequences of human Nav1.7, β1 and β2 are Q15858-3, Q07699 and O60939, respectively. The accession codes for the coordinates of Nav1.7, Nav1.5-Flecainide, Nav1.5-Propafenone and Nav1.5-Qunidine used in this study are 6J8J, 6UZ0, 7FBS and 6LQA, respectively. The accession code for the EM map of Nav1.7 used in this study is EMD-9782. The three-dimensional cryo-EM density maps of the human NaV1.7-β1-β2–XEN907, NaV1.7-β1-β2–TC-N1752 and NaV1.7-β1-β2–NaV1.7-IN2 have been deposited in the Electron Microscopy Database under accession codes EMD-33292, EMD-33295 and EMD-33296, respectively. The coordinates of the NaV1.7-β1-β2–XEN907, NaV1.7-β1-β2–TC-N1752 and NaV1.7-β1-β2–NaV1.7-IN2 have been deposited in the PDB under accession codes 7XM9, 7XMF and 7XMG, respectively. Source data are provided with this paper.

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Acknowledgements

We thank X. Huang, B. Zhu, X. Li, L. Chen and other staff members at the Center for Biological Imaging, Core Facilities for Protein Science at the Institute of Biophysics, Chinese Academy of Science (IBP, CAS) and D. Sun at the SM10 Cryo-EM Facility at the Institute of Physics, Chinese Academy of Sciences (IOP, CAS) for the support in cryo-EM data collection. We thank X.C. Zhang and Y. Zhao for their helpful discussions, X. Cui, Y. Dong, Z. Yu, Y. Wu and W. Fan for their research assistant service. This work is funded by Institute of Physics, Chinese Academy of Sciences (grant nos. E0VK101 and E2V4101 to D.J.), the National Natural Science Foundation of China (grant nos. T2221001 and 32271272 to D.J., 31871083 and 82271498 to Z.H. and 82071851 to J.G.), the Chinese Academy of Sciences Strategic Priority Research Program (grant nos. 31871083 and 81371432 to Z.H.), the Chinese National Programs for Brain Science and Brain-like intelligence technology (grant no. 2021ZD0202102 to Z.H.) and the program for the HUST Academic Frontier Youth Team (grant no. 5001170068 to J.G.).

Author information

Authors and Affiliations

Authors

Contributions

D.J. designed the experiments. J.Z., Y.L. and B.Y. prepared sample for cryo-EM study and made all the constructs. J.Z. and D.J. collected cryo-EM data. D.J. processed the data, built and refined the models. J.Z. and Y.S. prepared figures. Y.S. and Z.H. collected the electrophysiology data. J.G., Z.H. and D.J. analyzed and interpreted the results. J.Z., Y.S. and D.J. wrote the paper, and all authors reviewed and revised the paper.

Corresponding author

Correspondence to Daohua Jiang.

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Nature Structural & Molecular Biology thanks Vladimir Yarov-Yarovoy and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available. Primary Handling Editor: Florian Ullrich, in collaboration with the Nature Structural & Molecular Biology team.

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Extended data

Extended Data Fig. 1 Functional characterizations and purification of NaV1.7.

a. Functional characterizations of human WT NaV1.7. Normalized conductance-voltage (G/V) relationship (Red) and steady-state fast inactivation (Blue) of NaV1.7. A schematic diagram of the recording protocol is presented below representative current traces. The Boltzmann distribution has been fitted to each data set, yielding voltage-dependence of activation V1/2 = −20.7 ± 0.7 mV (n = 13) and steady-state fast inactivation V1/2 = −76.6 ± 0.9 mV (n = 14). Data are mean + /− s.e.m. b. A representative size exclusion chromatogram profile of purified NaV1.7-β1-β2 complex. Peak fractions between green dashed lines were collected and concentrated for cryo-EM study. c. The purified NaV1.7-β1-β2 complex sample was stained with Coomassie blue on SDS-PAGE gel. NaV1.7 and β1 are labelled by red arrows. The purification was repeated independently for more than 4 times with consistent results. Source data are provided.

Source data

Extended Data Fig. 2 Cryo-EM data processing of NaV1.7-antagonist complexes.

a. The workflow of cryo-EM data processing. A total of 2,900, 2,601 and 4,430 movie stacks were collected for NaV1.7-β1-β2-XEN907 (orange), TC-N1752 (blue), and NaV1.7-IN2 (pink) respectively. Particles were auto-picked in Relion3, 2D and 3D classifications were conducted to remove bad particles, followed by 3D AutoRefine in Relion3. Subsequent Polish and CTF Refine improved image quality. According to the gold-standard Fourier Shell Correlations (FSC) criterion, the final maps were determined to 3.22, 3.09, and 3.07 Å, respectively. b-d, Sharpened map of the NaV1.7XEN (b), NaV1.7TCN (c) and NaV1.7IN2 (d) complex, colored according to the local resolution values (left). Particle angular distribution for the final 3D reconstruction (middle). FSC of the final map of the complex, calculated between two independently refined half-maps before (blue) and after (red) post-processing, overlaid with an FSC curve calculated between the cryo-EM density map and the structural model shown in black (right).

Extended Data Fig. 3 EM map of the NaV1.7XEN.

a. The EM map of the S1-S6 segment in each domain. b. The EM map for P-loop of each domain. c. The auxiliary β1 and β2 in the NaV1.7 complex are shown individually. Side- chains of residues with good density are shown in sticks. The maps were prepared in PyMOL.

Extended Data Fig. 4 The overall structure of the drug-binding complex of human NaV1.7.

a. The EM map and model of the NaV1.7 complex viewed in parallel to the membrane plane. The β1 and β2 subunits, N-terminus domain (NTD), extracellular loops (ECLs), domain III and domain IV linker (LinkerIII-IV), and transmembrane helices S1–S6 of domain I were labeled. The α subunit was colored in cyan (DI), light red (DII), light green (DIII), light blue (DIV), and pink (LinkerIII-IV), respectively. The β1, β2 N-glycans and lipids were colored in wheat, light gray, yellow and orange, respectively. b. Overall structure comparison of human NaV1.7 α subunit-XEN907/TC-N1752/NaV1.7-IN2. The overall structures of each α subunit were colored in yellow (XEN907), blue (TC-N1752) and light pink (NaV1.7-IN2). c. Ion conductance path of NaV1.7 calculated by HOLE. The three panels on the left represent the pore radius of NaV1.7 complexed with XEN907, TC-N1752 and NaV1.7-IN2, respectively. The ligands in the cavity were omitted when calculating the pore radius. The selective filter (SF) and the intracellular activation gate (AG) were highlighted in pink and blue, respectively.

Extended Data Fig. 5 The NTD modeling and sequence alignments among human NaV channels.

a. The un-sharpened and sharpened (using DeepEMhancer) EM maps of NaV1.7XEN. b. The EM map of the N-terminus domain (NTD) was shown in surface, the secondary structure α1, α2, β1, β2 and amino acids with good density were displayed and labeled. c. AlphaFold2 model of NaV1.7 NTD. d. Structural comparison of the NaV1.7 NTD and its AlphaFold2 model. e. Gain-of-function, loss-of-function and other pathogenic mutation sites in the NTD of NaV channels are highlighted in green, red and gray, respectively. Secondary structure elements are indicated on top of the sequences. R116 in S0I of NaV1.7 mediates key interactions between the NTD and VSDI is identical among the isoforms.

Extended Data Fig. 6 Modulation of NaV1.7 by XEN907.

a. State-dependent inhibition of NaV1.7 by XEN907. A family of representative sodium currents in response to repetitive test pulses in the presence of 100 nM XEN907 when holding at −80 mV (Red) and −120 mV (Blue), respectively. b. Use-dependent inhibition of NaV1.7 by XEN907. Normalized current in response to repetitive test pulses in the presence of varied concentrations of XEN907 when holding at −80 mV (Red) and −120 mV (Blue), respectively. Data are mean + /− s.e.m. The n values of control, 0.01 nM, 0.1 nM, 1 nM, 10 nM, 100 nM for holding at −80 mV are 4, 3, 6, 5, 5, 4; and the n values of control, 1 nM, 10 nM, 100 nM, 1 μM, 10 μM for holding at −120 mV are 4, 7, 5, 5, 5, 4, respectively. c. The effect of 100 nM XEN907 on the voltage-dependence of activation. Data are mean + /− s.e.m. The n values for control (black) and XEN907 (red) are 6 and 8 respectively. d. The concentration-dependent shift of voltage-dependence of fast inactivation by XEN907. Data are mean + /− s.e.m. The n values of tested concentrations of control, 1 nM, 100 nM, and 10 μM are 9, 8, 12, 6, respectively. e. The EM density of XEN907. f. Pore density of NaV1.7apo (EMDB access code: EMD-9782) and NaV1.7XEN. The EM density for XEN907 is highlighted in yellow. g. EM densities S6IV of NaV1.7XEN and NaV1.7apo (PDB code: 6J8J). h. Mutants N1753M and M1754N are non-functional. Source data are provided.

Source data

Extended Data Fig. 7 Modulation of NaV1.7 by TC-N1752.

a. State-dependent inhibition of NaV1.7 by TC-N1752. A family of sodium currents in response to repetitive test pulses in the presence of 1 μM TC-N1752 when holding at −80 mV (Red) and −120 mV (Blue), respectively. b. Use-dependent inhibition of NaV1.7 by TC-N1752. Normalized current in response to repetitive test pulses in the presence of varied concentrations of TC-N1752 when holding at −80 mV (Red) and −120 mV (Blue), respectively. Data are mean + /− s.e.m. The n values of control, 0.01 μM, 0.03 μM, 0.1 μM, 0.3 μM, 1 μM, 10 μM for holding at −80 mV are 6, 4, 4, 6, 4, 4, 4; and for holding at −120 mV are 3, 4, 3, 3, 4, 5, 5, respectively. c-d. Use-dependent inhibition of NaV1.5 by TC-N1752. Data are mean + /− s.e.m. The n values of control, 1 nM, 10 nM, 0.1 μM, 1 μM, 10 μM are 3, 5, 5, 7, 10, 6, respectively. e. The dose-dependent response curve of TC-N1752 holding at −80 mV. Data are mean + /− s.e.m. The n values of 1 nM, 10 nM, 0.1 μM, 1 μM, 10 μM are 5, 4, 6, 9, 4, respectively. f. The effect of 10 μM TC-N1752 on the voltage-dependence of activation. Data are mean + /− s.e.m. The n values for control (black) and TC-N1752 (red) are 11 and 10 respectively. g. The EM density of TC-N1752. h. Pore density of NaV1.7apo (EMDB access code: EMD-9782) and NaV1.7TCN. The EM density for TC-N1752 is highlighted in blue. i. EM density for S6II of NaV1.7TCN and NaV1.7apo (PDB code: 6J8J). Source data are provided.

Source data

Extended Data Fig. 8 Modulation of NaV1.7 by NaV1.7-IN2.

a. State-independent inhibition of NaV1.7 by NaV1.7-IN2. A family of sodium currents in response to repetitive test pulses in the presence of 1 μM NaV1.7-IN2 when holding at −80 mV (Red) and −120 mV (Blue), respectively. b. Use-dependent inhibition of NaV1.7 by NaV1.7-IN2. Normalized current in response to repetitive test pulses in the presence of varied concentrations of NaV1.7-IN2 when holding at −80 mV (Red) and −120 mV (Blue), respectively. Data are mean + /− s.e.m. The n values of control, 0.1 nM, 1 nM, 10 nM, 0.1 μM, 1 μM for holding at −80 mV are 3, 3, 5, 3, 4, 3; and for holding at −120 mV are 3, 4, 3, 3, 3, 3, respectively. c, d. Use-dependent inhibition of NaV1.5 by NaV1.7-IN2. Data are mean + /− s.e.m. The n values of control, 0.1 nM, 1 nM, 10 nM, 0.1 μM, 1 μM are 4, 4, 5, 6, 4, 4, respectively. e. The dose-dependent response curve of NaV1.7-IN2 holding at −80 mV. Data are mean + /− s.e.m. The n values of 0.1 nM, 1 nM, 10 nM, 0.1 μM, 1 μM are 4, 8, 7, 4, 4, respectively. f. The effect of 100 nM NaV1.7-IN2 on the recovery from fast activation of NaV1.7. Data are mean + /− s.e.m. The n values for control (black) and NaV1.7-IN2 (red) are 8 and 7 respectively. g. The EM density of NaV1.7-IN2. h. Pore density of NaV1.7apo (EMDB access code: EMD-9782) and NaV1.7IN2. The EM density for NaV1.7-IN2 is highlighted in pink. Source data are provided.

Source data

Extended Data Fig. 9 The sequence alignments of P-loops and S6 helices of human NaV channels.

Residues that contribute to drug binding were shaded and labeled. Specifically, three NaV1.7 antagonists (XEN907, TC-N1752 and NaV1.7-IN2) and three previously reported anti-arrhythmic drugs (flecainide, PDB code: 6UZ0; propafenone, PDB code: 7FBS; quinidine, PDB code: 6LQA), were represented in different shapes and colors. The interacting residues were labeled with corresponding shapes. Residues interacting with the six blockers within 5 Å are highlighted.

Supplementary information

Reporting Summary

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Supplementary Video 1

XEN907 binding induced conformational changes in NaV1.7. XEN907 binds to the central cavity of NaV1.7, the binding directly causes an α- to π-helix transition of DIV-S6 helix, which tightens the fast inactivation gate.

Supplementary Video 2

TC-N1752 binding induced conformational changes in NaV1.7. TC-N1752 binds to the central cavity of NaV1.7, the binding indirectly causes an α- to π-helix transition of DII-S6 helix, which closes the activation gate.

Source data

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Zhang, J., Shi, Y., Huang, Z. et al. Structural basis for NaV1.7 inhibition by pore blockers. Nat Struct Mol Biol 29, 1208–1216 (2022). https://doi.org/10.1038/s41594-022-00860-1

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