Structural basis for high-voltage activation and subtype-specific inhibition of human Na v 1.8

Fig. 1.. Cryo-EM analysis of human Nav1.8 bound to selective blocker A-803467. (A) Inhibition of Nav1.8 by A-803467. Peak currents of Nav1.8 with A-803467 applied at different concentrations were recorded. Data represent mean ± SEM n = 5, 11, 13, 15, 6, and 6 for A-803467 concentrations at 10 nM, 100 nM, 300 nM, 1 μM, 10 μM, and 30 μM, respectively. Experimental details are presented in SI Appendix, Fig. S2A and Table S1 and Materials and Methods. (B) Heat map for resolution distribution of class I 3D EM reconstitution for human Nav1.8 in the presence of A-803467. Local resolutions were estimated with RELION 3.0 and represented in Chimera (53, 56). (C) Cryo-EM maps of three classes of VSDI. For visual clarity, the identical region of the map is colored gray and the three conformers of VSDI are colored as indicated. (D) VSDI of Nav1.8 was resolved in different conformations. As the resolution of the third class is insufficient for reliable side-chain assignment, we will focus on classes I and II for structural analysis. For visual clarity, sugar moieties, lipids, and a detergent molecule that penetrates the intracellular gate are not shown. All structure figures were prepared with Chimera…

Fig. 2.. Distinct conformations of VSDI. (A) Domainwise rotation of VSDI between the two classes. (Left) An intracellular view of the superimposed Nav1.8 structures, which deviate only in VSDI. (Middle and Right) Enlarged views of VSDI superimposed in the context of the overall structures. The PD segments in repeat II, which are identical in the two classes, are colored green. (B) Intradomain structural shifts between the two classes of VSDI. When compared individually, the two VSDI structures differ dramatically with a root-mean-square deviation of 2.8 Å over 88 Cα atoms. Gating charge residues, R2–K5, and the conserved residues on S2 and S3 that facilitate charge transfer are shown as thick sticks. Cα atoms of these residues are shown as spheres. (C) VSDI exhibits depolarized (up) conformation in both classes. The R2–R4 gating charge residues are above the occluding Tyr164 in both classes. The potential polar interactions, direct or indirect, are indicated by dashed lines. (D) S4I in class I has more extensive interactions with the neighboring S5II than that in class II. The interface residues between S4I and S5II are shown as sticks. (E) Altered interface between S1I and S5II in the…

Fig. 3.. The interface between VSDI and S5II in part accounts for the high-voltage activation of Nav1.8. (A) Structures of the transmembrane domains of human Nav1.8 and Nav1.7 mainly deviate in VSDI. Shown here is an extracellular view of the superimposed structures of Nav1.7 (PDB ID code: 7W9K) and the two classes of Nav1.8. ECLs are omitted for clarity. (B) Nav1.8-unique residues on S4I and S5II. Several S4I–S5II interface residues are invariant in all Nav subtypes except Nav1.8. These residues are shaded yellow. Refer to SI Appendix, Fig. S1 for complete sequence alignment of the four repeats of the nine human Nav channels. (C) Less-conserved VSDI–S5II interface between Nav1.8 and Nav1.7. Varied residues between Nav1.8 and Nav1.7 on the VSDI–S5II interface are shown as sticks. Shown here are two opposite side views to highlight the interface residues between S1I and S5II (Left) and between S4I and S5II (Right). The structures are superimposed as in A. (D) The Nav1.8-unique residues on the interface of VSDI and S5II are in part responsible for its dependence on more depolarized voltage of activation. Swapping the corresponding VSDI–S5II interface residues between Nav1.8 and Nav1.7 led…

Fig. 4.. Structural basis for specific blockade of Nav1.8 by A-803467. (A) A-803467 clenches S6IV of Nav1.8 beneath the selectivity filter. (Inset) Coordination of A-803467, shown as brown ball and sticks, by pore residues. Direct and water-mediated hydrogen bonds (H-bonds) are indicated by red and black dashed lines, respectively. (Right) Plane diagram of residues constituting the binding pocket within the cutoff distance from the ligand of 6 Å. The binding pocket and H-bond are indicated by gray dashed contour and red dashed lines, respectively. (B) The only change in the PD between the apo and A-803467 bound Nav1.8 structures is the swing of the side chain of Met1713. Shown on the left is an extracellular view of the superimposed PD of the apo (silver) and A-803467 bound (domain colored) structures of Nav1.8. (C) The only Nav1.8-unique blocker-binding residue Met1713 is not responsible for the subtype specificity. Among the Nav1.8 single-point mutants that were designed to validate the observed ligand binding, Ala substitution of Gln355, Phe386, and Thr1659 resulted in dramatically reduced peak currents, which were too small to support reliable analysis of pore blockage. Refer to SI…

Fig. 5.. The ECLs above the PD may be exploited for the development of Nav1.8-specific suppressors. (A) The ECLs exhibit marked structural differences between Nav1.8 and Nav1.7. Shown here is a tilted extracellular view of the superimposed overall structures of class I Nav1.8 (blue) and Nav1.7 (gray) (PDB ID code: 7W9K). The structural distinctions of the ECL above the PD are highlighted with oval shades. (B) Nav1.8 and Nav1.5, both TTX-resistant subtypes, also deviate substantially in the ECL region. Shown here is an extracellular view of the ECLs from the superimposed overall structures of human Nav1.8 and Nav1.5 (PDB ID code: 6LQA). (C) The different structures of the ECLs underlie the distinct sensitivities of Nav1.8 and Nav1.2 to the peptide pore blocker µ-conotoxin KIIIA (KIIIA). (Inset) An extracellular view of the ECLs in the superimposed structures of Nav1.8 and KIIIA-bound Nav1.2 (PDB ID code: 6J8E). The elongated ECLI in Nav1.8 may interfere with KIIIA binding.