Structural basis of Nav1.7 inhibition by an isoform-selective small-molecule antagonist.

INTRODUCTION Voltage-gated sodium (Nav) channels open and close ion-selective pores in response to changes in membrane potential, and this gating underlies the generation of action potentials. Nav channels are large membrane proteins that contain four peripheral voltage-sensor domains (VSD1–4) that influence the functional state of the central ion-conducting pore. Mutations within the nine human Nav channel isoforms are associated with migraine (Nav1.1), epilepsy (Nav1.1–Nav1.3, Nav1.6), pain (Nav1.7–Nav1.9), cardiac (Nav1.5), and muscle paralysis (Nav1.4) syndromes. Accordingly, Nav channel blockers are used for the treatment of many neurological and cardiovascular disorders. These drugs bind within the central pore domain and generally lack isoform selectivity owing to the high sequence conservation found among Nav channels, limiting their therapeutic utility. In this study, we focused on a recently identified class of isoform-selective small-molecule antagonists that target a unique binding site on the fourth voltage-sensor domain, VSD4. Here we report the structural determination of such small-molecule aryl sulfonamide antagonists in complex with human Nav1.7 VSD4. Our studies demonstrate how this important new class of gating modifier engages VSD4 to inhibit Nav channel activity through a “voltage-sensor trapping” mechanism. RATIONALE For structural studies, we devised a novel protein-engineering strategy that overcomes the technical complexities of producing full-length human Nav channels. Exploiting the evolutionary relationship between human and bacterial Nav channels, we fused portions of Nav1.7 VSD4 onto the bacterial channel NavAb. Using ligand-binding assays and alanine-scanning mutagenesis, we demonstrated that the antagonist binding site present in the human Nav1.7 channel is preserved within this human VSD4-NavAb chimeric channel. This chimeric construct allowed purification, crystallization, and structure determination of potent aryl sulfonamide antagonists in complex with the human Nav1.7 VSD4 binding site. RESULTS Functional studies using patch-clamp electrophysiology revealed that aryl sulfonamide inhibitors bind with high affinity to an isoform-selective and extracellularly accessible site on VSD4. These inhibitors show a high level of state dependence, potently blocking human Nav1.7 only when VSD4 is in its activated conformation. Our crystallographic studies revealed that the anionic warhead from the aryl sulfonamide inhibitors directly engages the fourth gating charge residue (R4) on the voltage-sensing S4 helix, effectively trapping VSD4 in its activated state. Isoform selectivity is achieved by inhibitor interactions with nonconserved residues found on the S2 and S3 transmembrane helices. The drug receptor site is partially submerged within the membrane bilayer, and a peripherally bound phospholipid was observed to form a tripartite complex with the antagonist and channel. CONCLUSION A new crystallization strategy has enabled the structural determination of VSD4 from human Nav1.7 in complex with potent, state-dependent, isoform-selective small-molecule antagonists. Mechanistically, inhibitor binding traps VSD4 in an activated conformation, which stabilizes a nonconductive state of the channel, and likely prevents recovery from inactivation. Unique phospholipid interactions and an exposed inhibitor binding site expand the importance of the membrane bilayer in ion channel biology. We anticipate that these structures will enable drug design efforts aimed at other voltage-gated ion channels and may accelerate the development of new treatments for pain that selectively target Nav1.7.