Structure of the human voltage-gated sodium channel Nav1.4 in complex with ß1.

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-ß1 complex at 3.2-Å resolution. Accurate model building was made for the pore domain, the voltage-sensing domains, and the ß1 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.

Parallel homodimer structures of the extracellular domains of the voltage-gated sodium channel ß4 subunit explain its role in cell-cell adhesion.

Voltage-gated sodium channels (VGSCs) are transmembrane proteins required for the generation of action potentials in excitable cells and essential for propagating electrical impulses along nerve cells. VGSCs are complexes of a pore-forming α subunit and auxiliary ß subunits, designated as ß1/ß1B-ß4 (encoded by SCN1B-4B, respectively), which also function in cell-cell adhesion. We previously reported the structural basis for the trans homophilic interaction of the ß4 subunit, which contributes to its adhesive function. Here, using crystallographic and biochemical analyses, we show that the ß4 extracellular domains directly interact with each other in a parallel manner that involves an intermolecular disulfide bond between the unpaired Cys residues (Cys58) in the loop connecting strands B and C and intermolecular hydrophobic and hydrogen-bonding interactions of the N-terminal segments (Ser30-Val35). Under reducing conditions, an N-terminally deleted ß4 mutant exhibited decreased cell adhesion compared with the wild type, indicating that the ß4 cis dimer contributes to the trans homophilic interaction of ß4 in cell-cell adhesion. Furthermore, this mutant exhibited increased association with the α subunit, indicating that the cis dimerization of ß4 affects α-ß4 complex formation. These observations provide the structural basis for the parallel dimer formation of ß4 in VGSCs and reveal its mechanism in cell-cell adhesion.

Structure-based site-directed photo-crosslinking analyses of multimeric cell-adhesive interactions of voltage-gated sodium channel ß subunits.

The ß1, ß2, and ß4 subunits of voltage-gated sodium channels reportedly function as cell adhesion molecules. The present crystallographic analysis of the ß4 extracellular domain revealed an antiparallel arrangement of the ß4 molecules in the crystal lattice. The interface between the two antiparallel ß4 molecules is asymmetric, and results in a multimeric assembly. Structure-based mutagenesis and site-directed photo-crosslinking analyses of the ß4-mediated cell-cell adhesion revealed that the interface between the antiparallel ß4 molecules corresponds to that in the trans homophilic interaction for the multimeric assembly of ß4 in cell-cell adhesion. This trans interaction mode is also employed in the ß1-mediated cell-cell adhesion. Moreover, the ß1 gene mutations associated with generalized epilepsy with febrile seizures plus (GEFS+) impaired the ß1-mediated cell-cell adhesion, which should underlie the GEFS+ pathogenesis. Thus, the structural basis for the ß-subunit-mediated cell-cell adhesion has been established.

Navß4 regulates fast resurgent sodium currents and excitability in sensory neurons.

BACKGROUND: Increased electrical activity in peripheral sensory neurons including dorsal root ganglia (DRG) and trigeminal ganglia neurons is an important mechanism underlying pain. Voltage gated sodium channels (VGSC) contribute to the excitability of sensory neurons and are essential for the upstroke of action potentials. A unique type of VGSC current, resurgent current (INaR), generates an inward current at repolarizing voltages through an alternate mechanism of inactivation referred to as open-channel block. INaRs are proposed to enable high frequency firing and increased INaRs in sensory neurons are associated with pain pathologies. While Nav1.6 has been identified as the main carrier of fast INaR, our understanding of the mechanisms that contribute to INaR generation is limited. Specifically, the open-channel blocker in sensory neurons has not been identified. Previous studies suggest Navß4 subunit mediates INaR in central nervous system neurons. The goal of this study was to determine whether Navß4 regulates INaR in DRG sensory neurons. RESULTS: Our immunocytochemistry studies show that Navß4 expression is highly correlated with Nav1.6 expression predominantly in medium-large diameter rat DRG neurons. Navß4 knockdown decreased endogenous fast INaR in medium-large diameter neurons as measured with whole-cell voltage clamp. Using a reduced expression system in DRG neurons, we isolated recombinant human Nav1.6 sodium currents in rat DRG neurons and found that overexpression of Navß4 enhanced Nav1.6 INaR generation. By contrast neither overexpression of Navß2 nor overexpression of a Navß4-mutant, predicted to be an inactive form of Navß4, enhanced Nav1.6 INaR generation. DRG neurons transfected with wild-type Navß4 exhibited increased excitability with increases in both spontaneous activity and evoked activity. Thus, Navß4 overexpression enhanced INaR and excitability, whereas knockdown or expression of mutant Navß4 decreased INaR generation. CONCLUSION: INaRs are associated with inherited and acquired pain disorders. However, our ability to selectively target and study this current has been hindered due to limited understanding of how it is generated in sensory neurons. This study identified Navß4 as an important regulator of INaR and excitability in sensory neurons. As such, Navß4 is a potential target for the manipulation of pain sensations.

Crystallographic insights into sodium-channel modulation by the ß4 subunit.

Voltage-gated sodium (Nav) channels are embedded in a multicomponent membrane signaling complex that plays a crucial role in cellular excitability. Although the mechanism remains unclear, ß-subunits modify Nav channel function and cause debilitating disorders when mutated. While investigating whether ß-subunits also influence ligand interactions, we found that ß4 dramatically alters toxin binding to Nav1.2. To explore these observations further, we solved the crystal structure of the extracellular ß4 domain and identified (58)Cys as an exposed residue that, when mutated, eliminates the influence of ß4 on toxin pharmacology. Moreover, our results suggest the presence of a docking site that is maintained by a cysteine bridge buried within the hydrophobic core of ß4. Disrupting this bridge by introducing a ß1 mutation implicated in epilepsy repositions the (58)Cys-containing loop and disrupts ß4 modulation of Nav1.2. Overall, the principles emerging from this work (i) help explain tissue-dependent variations in Nav channel pharmacology; (ii) enable the mechanistic interpretation of ß-subunit-related disorders; and (iii) provide insights in designing molecules capable of correcting aberrant ß-subunit behavior.

Antagonism of lidocaine inhibition by open-channel blockers that generate resurgent Na current.

Na channels that generate resurgent current express an intracellular endogenous open-channel blocking protein, whose rapid binding upon depolarization and unbinding upon repolarization minimizes fast and slow inactivation. Na channels also bind exogenous compounds, such as lidocaine, which functionally stabilize inactivation. Like the endogenous blocking protein, these use-dependent inhibitors bind most effectively at depolarized potentials, raising the question of how lidocaine-like compounds affect neurons with resurgent Na current. We therefore recorded lidocaine inhibition of voltage-clamped, tetrodotoxin-sensitive Na currents in mouse Purkinje neurons, which express a native blocking protein, and in mouse hippocampal CA3 pyramidal neurons with and without a peptide from the cytoplasmic tail of NaVß4 (the ß4 peptide), which mimics endogenous open-channel block. To control channel states during drug exposure, lidocaine was applied with rapid-solution exchange techniques during steps to specific voltages. Inhibition of Na currents by lidocaine was diminished by either the ß4 peptide or the native blocking protein. In peptide-free CA3 cells, prolonging channel opening with a site-3 toxin, anemone toxin II, reduced lidocaine inhibition; this effect was largely occluded by open-channel blockers, suggesting that lidocaine binding is favored by inactivation but prevented by open-channel block. In constant 100 µm lidocaine, current-clamped Purkinje cells continued to fire spontaneously. Similarly, the ß4 peptide reduced lidocaine-dependent suppression of spiking in CA3 neurons in slices. Thus, the open-channel blocking protein responsible for resurgent current acts as a natural antagonist of lidocaine. Neurons with resurgent current may therefore be less susceptible to use-dependent Na channel inhibitors used as local anesthetic, antiarrhythmic, and anticonvulsant drugs.