Molecular determinants of μ-conotoxin KIIIA interaction with the human voltage-gated sodium channel NaV1.7

The voltage-gated sodium (Nav) channel subtype Nav1.7 plays a critical role in pain signaling, making it an important drug target. A number of peptide toxins from cone snails (conotoxins) bind to the extracellular vestibule of the Nav channel pore and block ion conduction. While the known conotoxins have variable selectivity among Nav channel subtypes, they form potential scaffolds for engineering of selective and potent channel inhibitors. Here we studied the molecular interactions between μ-conotoxin KIIIA (KIIIA) and the human Nav1.7 channel (hNav1.7). Using the cryo-electron microscopy (cryo-EM) structure of the electric eel Nav1.4 channel as a template we developed a structural model of hNav1.7 with Rosetta computational modeling. We performed in silico docking of KIIIA using RosettaDock and identified residues forming specific pairwise contacts between KIIIA and hNav1.7. Pairwise interactions were experimentally validated using mutant cycle analysis. Comparison with a recently published cryo-EM structure of the KIIIA-hNav1.2 channel complex revealed key similarities and differences between channel subtypes with potential implications for the molecular mechanism of toxin block. Our integrative approach, combining high-resolution structural data with computational modeling and experimental validation, will be useful for engineering of molecular probes to study Nav channels function and for rational design of novel biologics to treat chronic pain, cardiac arrhythmias, and epilepsy.