A sodium channel inhibitor ISTX-I with a novel structure provides a new hint at the evolutionary link between two toxin folds

Abstract

Members of arachnida, such as spiders and scorpions, commonly produce venom with specialized venom glands, paralyzing their prey with neurotoxins that specifically target ion channels. Two well-studied motifs, the disulfide-directed hairpin (DDH) and the inhibitor cystine knot motif (ICK), are both found in scorpion and spider toxins. As arachnids, ticks inject a neurotoxin-containing cocktail from their salivary glands into the host to acquire a blood meal, but peptide toxins acting on ion channels have not been observed in ticks. Here, a new neurotoxin (ISTX-I) that acts on sodium channels was identified from the hard tick Ixodes scapularis and characterized. ISTX-I exhibits a potent inhibitory function with an IC50 of 1.6 μM for sodium channel Nav1.7 but not other sodium channel subtypes. ISTX-I adopts a novel structural fold and is distinct from the canonical ICK motif. Analysis of the ISTX-I, DDH and ICK motifs reveals that the new ISTX-I motif might be an intermediate scaffold between DDH and ICK, and ISTX-I is a clue to the evolutionary link between the DDH and ICK motifs. These results provide a glimpse into the convergent evolution of neurotoxins from predatory and blood-sucking arthropods.

Figure 1. Determination of the disulfide bond pattern in ISTX-I.

(A) RP-HPLC chromatogram of the ISTX-I reaction mixture after partial reduction by TCEP. (B) The sequencing maps for the Edman degradation phenylthiohydantoins observed on cycles 2, 8, 13, 14, and 28 of the two thiol-reduced and carboxyamidomethylated peaks 3. (C) The sequencing maps for the Edman degradation phenylthiohydantoins observed oncycles 2, 8, 13, 14 and 28 of the two thiol-reduced and carboxyamidomethylated peaks 2. (D) The MALDI-TOF-MS analysis of the two thiol-reduced and carboxyamidomethylated peaks after enzyme digestion.

Figure 2. Three-dimensional structure of ISTX-I, with the yellow ball-and-stick format representing the disulfide bond connectivity (C₁-C₄, C₂-C₅, and C₃-C₆).

(A) Backbone traces from the structural ensemble of ISTX-I with the well-refined region in red and the terminal disorder loops in gray. (B) Cartoon diagram of ISTX-I illustrating the locations of the secondary structures. The cysteine residues are labeled with Roman numerals. The β-strands are colored green and others are in gray. (C) The structure of Huwentoxin-IV demonstrates the canonical ICK fold (pdb code: 1MB6). (D) Superposition of the structures of ISTX-I (green), ICK fold Huwentoxin-IV (yellow) and the DDH fold delta-atracotoxin-HV1(1VTX) (cyan) with the two beta sheets. The disulfide bonds are labeled and color-coded to demonstrate the differences between the three different folds. (E) Sequence alignment of the selected ICK, ISTX-I and DDH motifs. The disulfide bonds in ICK motifs are described above the sequence. The positions of the disulfide bonds in DDH motifs are colored yellow below the sequences.

Figure 3. Effects of ISTX-I on voltage-gated ion channels in rat DRG neurons.

(A) Inhibition of TTX-s Nav channel currents by 1 μM ISTX-I. (B) Effects of 10 μM ISTX-I on TTX-r Nav channel currents. (C) Dose-dependent inhibition of ISTX-I on the TTX-s Nav channel (n = 5). (D) Current-voltage (I-V) relationship for the TTX-s Nav channel currents before (solid circles) and after (open circles) the application of 5 μM ISTX-I (n = 5). (E) Conductance–voltage (G-V) relationship of the TTX-s Nav channel before (solid circles) and after (open circles) treatment with 5 μM ISTX-I (n = 5). (F) Steady-state inactivation of the TTX-s Nav channel currents before (solid circles) and after(open circles) the application of 10 μM ISTX-I (n = 5).

Figure 4. Effects of ISTX-I expressed in HEK293 cells.

Current traces were evoked with a 50ms step depolarization to −10 mV from a holding potential of −80 mV every 5s. (A) Inhibition of hNav1.7 by 1 μM ISTX-I. (B) Dose-dependent inhibition of the hNav1.7 Nav channel by ISTX-I. Notably, 5 μM ISTX-I had no effect on the current-voltage (I-V) relationships (C) or on the conductance–voltage (G-V) relationships (D) of hNav1.7. (E) Steady-state inactivation of the hNav1.7 channel before (solid circles) and after (open circles) the application of 10 μM ISTX-I (n = 5).

Figure 5. Phylogenetic analysis of ISTX-I with spider and scorpion toxins.

Phylogenetic dendrogram obtained with a neighbor-joining analysis based on the proportion difference (p-distance) of aligned amino acids of the full-length peptide sequences. All of the spider and scorpion toxin sequences are from UniProt.