Cryo-EM structure of human voltage-gated sodium channel Nav1.6

Abstract

Voltage-gated sodium channel Na_v1.6 plays a crucial role in neuronal firing in the central nervous system (CNS). Aberrant function of Na_v1.6 may lead to epilepsy and other neurological disorders. Specific inhibitors of Na_v1.6 thus have therapeutic potentials. Here we present the cryo-EM structure of human Na_v1.6 in the presence of auxiliary subunits β1 and fibroblast growth factor homologous factor 2B (FHF2B) at an overall resolution of 3.1 Å. The overall structure represents an inactivated state with closed pore domain (PD) and all "up" voltage-sensing domains. A conserved carbohydrate-aromatic interaction involving Trp302 and Asn326, together with the β1 subunit, stabilizes the extracellular loop in repeat I. Apart from regular lipids that are resolved in the EM map, an unprecedented Y-shaped density that belongs to an unidentified molecule binds to the PD, revealing a potential site for developing Na_v1.6-specific blockers. Structural mapping of disease-related Na_v1.6 mutations provides insights into their pathogenic mechanism.

Keywords: Nav1.6; cryo-EM; epilepsy; ion channel.

Fig. 1.

Cryo-EM structure of human Na_v1.6-β1. (A) Electrophysiological properties of Na_v1.6 alone or co-expressed with the auxiliary subunits β1 and FHF2B in HEK293T cells. Shown here are the voltage-dependent activation and inactivation curves. Please refer to Methods and SI Appendix, Table S1 for details. (B) Cryo-EM map of the Na_v1.6 complex comprising the α1 (domain colored) and β1 (light salmon) subunits. The cytosolic III-IV linker is colored chocolate. Sugar moieties and lipids are colored gray and orange, respectively. The same color scheme is applied throughout the manuscript. (C) Overall structure of the Na_v1.6 complex in a Side view (Left) and a cytoplasmic view (Right). Sugar moieties are shown as sticks, and the IFM motif within the cytosolic III-IV linker is shown as spheres.

Fig. 2.

ECL_I is sandwiched by β1 and the glycan attached to Asn326. (A) ECLs are the least conserved structural elements among Na_v subtypes. The conservation scores (calculated by ConSurf) of human Na_vs are color-coded and presented on the Na_v1.6 structure. The ECL_I and ECL_III region are shown as surface. Two interfaces, ECL_I-β1 and ECL_I-glycan, are highlighted in squares. (B) Extensive interactions between ECL_I and β1. Residues forming the hydrogen bond network, indicated by red, dashed lines, at the interface of ECL_I and β1 are shown. (C) Interactions between ECL_I and Asn326-glycan. Two aromatic residues, Trp302 and Phe288, are involved in the carbohydrate–aromatic interactions. The density in the Left panel is contoured at 5.2 σ. (D) Multiple sequence alignment of ECL_I in human Na_v subtypes. The alignment, calculated in Clustal X, is shaded in different colors based on sequence conservation and the chemical properties of the residues. The conserved glycosylation site (Asn326 in Na_v1.6) is indicated. (E) Schematic illustration of the chemical assembly of the branched N-GlcNAc₂Man₇ glycan. Related key residues are labeled in blue ovals. (F) Density variations of the corresponding glycan in different Na_v subtypes. Red arrows indicate the observed GlcNAc₂ and GlcNAc in Na_v1.5 (EMD-30850) and Na_v1.8 (EMD-32439), respectively.

Fig. 3.

Unique ligand density in the inactivated structure of Na_v1.6. (A) An unidentified density bound to the IV-I fenestration of the PD. A Side view and a Top view of the PD are shown. A putative PE is shown as gray ball-and-sticks in the semitransparent gray density. The density-colored pale yellow may belong to a molecule whose identity is unknown. (B) The intracellular gate of Na_v1.6 is penetrated by a GDN (green spheres). The permeation path of Na_v1.6 is calculated in HOLE, and the pore radii are superimposed with that of human Na_v1.2 (green) and Na_v1.7 (pink) (43). (C) VSD_IV exhibits a slightly different conformation in different Na_v channels. Structures of VSD_IV from Na_v1.6 (cyan), Na_v1.2 (green), and Na_v1.7 (pink) are superimposed relative to the overall structure. The PDB codes for Na_v1.2 and Na_v1.7 are 6J8E and 7W9K, respectively. Two perpendicular views are shown. (D) Distinct coordination of GC residues in VSD_IV of Na_v1.7 (Left) and Na_v1.6 (Right). The GC residues on S4_IV and their coordinating residues on S1_IV-S3_IV are shown as sticks, and the potential hydrogen bonds are shown as red-dashed lines.

Fig. 4.

Structural mapping of disease-related mutations in human Na_v1.6. (A) Mapping of the disease-associated mutations on the structure of Na_v1.6 in two perpendicular views. The Cα atoms of the disease-related residues are shown as spheres and colored for different types of disease. DEE13 (purple); BFIS5 (blue); MYOCL2 (red). (B) Mapping of the disease-related mutations in each domain of human Na_v1.6.