Crystallographic insights into sodium-channel modulation by the β4 subunit

Voltage-gated sodium (Na-v) channels are embedded in a multicomponent membrane signaling complex that plays a crucial role in cellular excitability. Although the mechanism remains unclear, beta-subunits modify Na-v channel function and cause debilitating disorders when mutated. While investigating whether beta-subunits also influence ligand interactions, we found that beta 4 dramatically alters toxin binding to Na(v)1.2. To explore these observations further, we solved the crystal structure of the extracellular beta 4 domain and identified (58)Cys as an exposed residue that, when mutated, eliminates the influence of beta 4 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 beta 4. Disrupting this bridge by introducing a beta 1 mutation implicated in epilepsy repositions the (58)Cys-containing loop and disrupts beta 4 modulation of Na(v)1.2. Overall, the principles emerging from this work (i) help explain tissuedependent variations in Nav channel pharmacology; (ii) enable the mechanistic interpretation of beta-subunit-related disorders; and (iii) provide insights in designing molecules capable of correcting aberrant beta-subunit behavior.