Research Article

Structure of the human voltage-gated sodium channel Nav1.4 in complex with β1

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Science  19 Oct 2018:
Vol. 362, Issue 6412, eaau2486
DOI: 10.1126/science.aau2486

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Structures of voltage-gated sodium channels

In “excitable” cells, like neurons and muscle cells, a difference in electrical potential is used to transmit signals across the cell membrane. This difference is regulated by opening or closing ion channels in the cell membrane. For example, mutations in human voltage-gated sodium (Nav) channels are associated with disorders such as chronic pain, epilepsy, and cardiac arrhythmia. Pan et al. report the high-resolution structure of a human Nav channel, and Shen et al. report the structures of an insect Nav channel bound to the toxins that cause pufferfish and shellfish poisoning in humans. Together, the structures give insight into the molecular basis of sodium ion permeation and provide a path toward structure-based drug discovery.

Science, this issue p. eaau2486, p. eaau2596

Structured Abstract


The nine subtypes of mammalian voltage-gated sodium (Nav) channels, Nav1.1 to Nav1.9, are responsible for the initiation and propagation of action potentials in specific excitable systems, among which Nav1.4 functions in skeletal muscle. Responding to membrane potential changes, Nav channels undergo sophisticated conformational shifts that lead to transitions between resting, activated, and inactivated states. Defects in Nav channels are associated with a variety of neurological, cardiovascular, muscular, and psychiatric disorders. In addition, Nav channels are targets for natural toxins and clinical therapeutics.

Understanding the physiological and pathophysiological mechanisms of Nav channels requires knowing the structure of each conformational state. All eukaryotic Nav 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 Nav 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 Nav 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 Nav1.4 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 Nav1.4-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 Nav 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, electromechanical coupling, asynchronous activation, and fast inactivation of the four repeats. It opens a new chapter for studying the structure-function relationships of Nav channels, affords an accurate template to map mutations associated with diseases such as myotonia and periodic paralysis hyperkalemic, and illuminates a path toward precise understanding and intervention with specific Nav channelopathies.

Structure of the human Nav1.4-β1 complex.

Two perpendicular views are shown. Left: Side view in ribbon cartoon. The VSDs are colored yellow, and the selectivity filter and supporting helices P1 and P2 are colored light cyan. The IFM motif is shown as spheres, and the III-IV linker is colored orange. The transmembrane segments in repeat IV are labeled. Right: Surface presentation for the bottom view to highlight the intracellular gate and the cavity that accommodates the IFM motif. The GDN molecule that penetrates the intracellular gate is shown as thin sticks.


Voltage-gated sodium (Nav) channels, which are responsible for action potential generation, are implicated in many human diseases. Despite decades of rigorous characterization, the lack of a structure of any human Nav channel has hampered mechanistic understanding. Here, we report the cryo–electron microscopy structure of the human Nav1.4-β1 complex at 3.2-Å resolution. Accurate model building was made for the pore domain, the voltage-sensing domains, and the β1 subunit, providing insight into the molecular basis for Na+ permeation and kinetic asymmetry of the four repeats. Structural analysis of reported functional residues and disease mutations corroborates an allosteric blocking mechanism for fast inactivation of Nav channels. The structure provides a path toward mechanistic investigation of Nav channels and drug discovery for Nav channelopathies.

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