Neurotoxins are of interest as lead molecules for the development of pharmaceuticals. Huwentoxin-IV (HwTx-IV) is a spider-venom peptide. This neurotoxin is one of the most potent human-NaV1.7 (hNaV1.7) inhibitors described to date. NaV1.7 is a subtype of voltage-gated sodium channel that is involved in the propagation of pain signals, and it is a validated analgesic target. HwTx-IV inhibits hNaV1.7 with an IC50 of ~20 nM but inhibits the cardiac subtype NaV1.5 with far lower potency. In this study, a rationally-designed, optimized, triple-mutant HwTx-IV analogue (oHwTx) was produced recombinantly via expression in the periplasm of E. coli. The mutant has significantly increased potency against hNaV1.7 (IC50 = 0.4 ± 0.1 nM) without increased potency against hNaV1.5. Neither its activity against other subtypes nor its structure has been characterised. Bacterial expression enabled production of uniformly 15N/13C-labelled recombinant peptide for structure determination using multidimensional heteronuclear NMR spectroscopy. Additionally the activity of the recombinant peptide on various NaV channel subtypes was measured via electrophysiology and FLIPR. In parallel the analgesic efficacy of the peptide was investigated using a rodent pain model. oHwTx and similar peptides from the same family inhibit NaV channel activation by binding to the voltage sensor (VSD) of domain-II (DII). Here, we applied NMR-based methods to probe the atomic details of the interaction between gating-modifier venom peptides and isolated DII-VSD of several hNaV subtypes. The knowledge gained from this work will lay the foundation for ligand-channel structural studies and provide a platform for rational engineering of subtype-selective inhibitors of hNaV1.7, as novel analgesics.