A hydrophobic loop of the spider-venom peptide Tl1a drives activity at NaV1.8

Voltage-gated sodium (NaVs) channels are pore-forming transmembrane proteins that regulate the influx of sodium ions across cell membranes. Spider venoms are a rich source of NaV-modulating peptides with high selectivity and potency, making them important tools for understanding NaV structure and function. NaV1.8 is tetrodotoxin-resistant, expressed in the peripheral nervous system and contributes to the propagation of action potentials in nociceptive neurons, making it a potential therapeutic target for pain. We identified Tl1a, a 36 amino acid residue peptide isolated from the crude venom of the Peruvian tarantula species Thrixopelma longicolli as a modulator of NaV1.8. Tl1a was synthesized using solid-phase peptide synthesis, and activity was assessed using automated whole-cell patch-clamp recordings. Tl1a inhibited NaV1.8 peak current (IC50 210 nM), delayed the kinetics of activation, inhibited fast inactivation, and caused a persistent current as well as a depolarising shift in the voltage dependence of activation (ΔV1/2 +11 mV). Tl1a inhibited peak current with similar potency at NaV1.5 (IC50 282 nM) and KV2.1 (IC50 156 nM) and was 8-fold selective over the tetrodotoxin-sensitive NaV1.4 (IC50 1769 nM), NaV1.1 (2201 nM) and 6-fold selective over NaV1.7 (IC50 1278 nM) channels. Tl1a analogues with an increased number of charged amino acids in loop 4 of the peptide lost activity at NaV1.8 due to altered interactions with the domain IV S3-S4 extracellular loop. The results of this work contribute to a better understanding of the structure-activity relationships at tetrodotoxin-resistant NaV channels and may be useful for the future rational design of selective NaV1.8 peptide modulators.

Keywords: Q2 2025