The nine subtypes of mammalian voltage-gated sodium (Na_v) channels, Na_v1.1 to Na_v1.9, are responsible for the initiation and propagation of action potentials in specific excitable systems, among which Na_v1.4 functions in skeletal muscle. Responding to membrane potential changes, Na_v channels undergo sophisticated conformational shifts that lead to transitions between resting, activated, and inactivated states. Defects in Na_v channels are associated with a variety of neurological, cardiovascular, muscular, and psychiatric disorders. In addition, Na_v channels are targets for natural toxins and clinical therapeutics.

Understanding the physiological and pathophysiological mechanisms of Na_v channels requires knowing the structure of each conformational state. All eukaryotic Na_v channels comprise a single polypeptide chain, the α subunit, that folds to four homologous repeats I to IV. Channel properties are modulated by one or two subtype-specific β subunits. Cryo–electron microscopy (cryo-EM) structures of two Na_v channels, one from American cockroach and the other from electric eel, were resolved in two distinct conformations. However, the inability to record currents of either channel in heterologous systems prevented functional assignment of these structures. Structural elucidation of a functionally well-characterized Na_v channel is required to establish a model for structure-function relationship studies.

After extensive screening for expression systems, protein boundaries, chimeras, affinity tags, and combination with subtype-specific β subunits, we focused on human Na_v1.4 in the presence of β1 subunit for cryo-EM analysis. The complex, which was transiently coexpressed in human embryonic kidney (HEK) 293F cells with BacMam viruses and purified through tandem affinity columns and size exclusion chromatography, was concentrated to ~0.5 mg/ml for cryo-EM sample preparation and data acquisition.

The cryo-EM structure of human Na_v1.4-β1 complex was determined to 3.2-Å resolution. The extracellular and transmembrane domains, including the complete pore domain, all four voltage-sensing domains (VSDs), and the β1 subunit, were clearly resolved, enabling accurate model building (see the figure).

The well-resolved Asp/Glu/Lys/Ala (DEKA) residues, which are responsible for specific Na⁺ permeation through the selectivity filter, exhibit identical conformations to those seen in the other two Na_v structures. A glyco-diosgenin (GDN) molecule, the primary detergent used for protein purification and cryo-EM sample preparation, penetrates the intracellular gate of the pore domain, holding it open to a diameter of ~5.6 Å. The central cavity of the pore domain is filled with lipid-like densities, which traverse the side wall fenestrations.

Voltage sensing involves four to six Arg/Lys residues on helix S4 of the VSD. This helix moves “up” (away from the cytoplasm) in response to changes of the membrane potential, and this opens the channel finally. All four VSDs display up conformations. The movement of the gating charge residues is facilitated by coordination to acidic and polar residues on S1 to S3. The improved resolution allows detailed analysis of the coordination.

The fast inactivation Ile/Phe/Met (IFM) motif on the short linker between repeats III and IV inserts into a hydrophobic cavity enclosed by the S6 and S4-S5 segments in repeats III and IV. Analysis of reported functional residues and disease mutations corroborates our recently proposed allosteric blocking mechanism for fast inactivation.

The structure provides important insight into the molecular basis for Na⁺ permeation …