Structural basis for inhibition of the voltage-gated sodium channel NaV1.7 by the tarantula toxin HWTX-I.

The voltage-gated sodium channel NaV1.7 is a pivotal therapeutic target for the developing novel, potent, and specific analgesics. HWTX-I, a peptide isolated from the venom of the tarantula Ornithoctonus huwena, is known to inhibit N-type calcium channels and has also been demonstrated to block tetrodotoxin-sensitive NaV channels in dorsal root ganglion neurons. In this study, we show that HWTX-I potently inhibits NaV1.7 currents with an IC50 value of 40.6 ± 16.1 nM, but it does not significantly affect the voltage dependence of NaV1.7 steady-state activation and inactivation. Alanine-scanning mutagenesis revealed that residues K3, V5, F6, P11, N14, E15, W28, and K30 are critical for HWTX-I's inhibitory activity against NaV1.7. Furthermore, site-directed mutagenesis analysis demonstrated that HWTX-I binds to the S3-S4 linker in domain II of NaV1.7, and the D816K mutation in NaV1.7 significantly abrogates the efficacy of the HWTX-I-NaV1.7 interaction. Molecular dynamics simulations combined with free energy decomposition analysis identified NaV1.7-D816 and HWTX-I-K3 as dominant energetic contributors to binding, consistent with a role for long-range electrostatic steering rather than a persistent short-range salt bridge. Collectively, these findings elucidate the structural basis of HWTX-I-NaV1.7 binding, identify the pharmacophore of the toxin, and provide valuable insights into the interactions between peptide toxins and NaV1.7. Therefore, this research may guide the future development of specific, safe, and efficacious NaV1.7 inhibitors for pain relief.