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

: Voltage-gated sodium (Na V s) channels are pore-forming transmembrane proteins that regulate the influx of sodium ions across cell membranes. Spider venoms are a rich source of Na V -modulating peptides with high selectivity and potency, making them important tools for understanding Na V structure and function. Na V 1.8 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 Na V 1.8. Tl1a was synthesized using solid-phase peptide synthesis, and activity was assessed using automated whole-cell patch-clamp recordings. Tl1a inhibited Na V 1.8 peak current (IC 50 210 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 (ΔV 1/2 +11 mV). Tl1a inhibited peak current with similar potency at Na V 1.5 (IC 50 282 nM) and K V 2.1 (IC 50 156 nM) and was 8-fold selective over the tetrodotoxin-sensitive Na V 1.4 (IC 50 1769 nM), Na V 1.1 (2201 nM) and 6-fold selective over Na V 1.7 (IC 50 1278 nM) channels. Tl1a analogues with an increased number of charged amino acids in loop 4 of the peptide lost activity at Na V 1.8 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 Na V channels and may be useful for the future rational design of selective Na V 1.8 peptide modulators.