Voltage-Gated Sodium Channel Modulation by a New Spider Toxin Ssp1a Isolated From an Australian Theraphosid

Abstract

Given the important role of voltage-gated sodium (NaV) channel-modulating spider toxins in elucidating the function, pharmacology, and mechanism of action of therapeutically relevant NaV channels, we screened the venom from Australian theraphosid species against the human pain target hNaV1.7. Using assay-guided fractionation, we isolated a 33-residue inhibitor cystine knot (ICK) peptide (Ssp1a) belonging to the NaSpTx1 family. Recombinant Ssp1a (rSsp1a) inhibited neuronal hNaV subtypes with a rank order of potency hNaV1.7 > 1.6 > 1.2 > 1.3 > 1.1. rSsp1a inhibited hNaV1.7, hNaV1.2 and hNaV1.3 without significantly altering the voltage-dependence of activation, inactivation, or delay in recovery from inactivation. However, rSsp1a demonstrated voltage-dependent inhibition at hNaV1.7 and rSsp1a-bound hNaV1.7 opened at extreme depolarizations, suggesting rSsp1a likely interacted with voltage-sensing domain II (VSD II) of hNaV1.7 to trap the channel in its resting state. Nuclear magnetic resonance spectroscopy revealed key structural features of Ssp1a, including an amphipathic surface with hydrophobic and charged patches shown by docking studies to comprise the interacting surface. This study provides the basis for future structure-function studies to guide the development of subtype selective inhibitors.

Structure-function and rational design of a spider toxin Ssp1a at human voltage-gated sodium channel subtypes

“…Fifteen rSsp1a alanine-analogues were produced via recombinant expression as previously described ( Dongol et al, 2021 ), and their activity tested on hNa V 1.2, hNa V 1.3 and hNa V 1.7 as a function of varying concentration ( Figure 1B ; Supplementary Figure S1 ; Supplementary Table S1 ). The alanine mutations causing ≥10-fold loss in potency at all three hNa V subtypes included W5A, F6A, W24A, K25A, Y26A, W28A and R30A.…”

“…rSsp1a analogues were recombinantly expressed in E. coli as a His 6 tagged-MBP fused peptide, harvested, and purified as described previously ( Dongol et al, 2021 ). Briefly, the mutant plasmids were transformed into BL21 (λDE3) competent E. coli cells and cultured in LB-Amp medium at 37°C, 120 rpm until the optical density at 600 nm (OD600) reached 0.8–1.0.…”

“…Ssp1a, a 33-residue inhibitor cystine knot (ICK) peptide ( Figure 1A ) belonging to the voltage-gated sodium channel modulator spider toxin family 1 (NaSpTx1), is a potent inhibitor of neuronal hNa V subtypes 1.7, 1.6, 1.3, 1.2 and 1.1 ( Dongol et al, 2021 ). The closest homologs with comprehensive structure-function data available are the distantly related GpTx-1 (44% identity) and HwTx-IV (40% identity), with HwTx-IV and recombinant Ssp1a (rSsp1a) showing similar pharmacology at hNa V 1.7 ( Xiao et al, 2008 ; Dongol et al, 2021 ).…”

“…Ssp1a, a 33-residue inhibitor cystine knot (ICK) peptide ( Figure 1A ) belonging to the voltage-gated sodium channel modulator spider toxin family 1 (NaSpTx1), is a potent inhibitor of neuronal hNa V subtypes 1.7, 1.6, 1.3, 1.2 and 1.1 ( Dongol et al, 2021 ). The closest homologs with comprehensive structure-function data available are the distantly related GpTx-1 (44% identity) and HwTx-IV (40% identity), with HwTx-IV and recombinant Ssp1a (rSsp1a) showing similar pharmacology at hNa V 1.7 ( Xiao et al, 2008 ; Dongol et al, 2021 ). While the NaSpTx1 toxin studies have focused on the development of hNa V 1.7-selective inhibitors ( Minassian et al, 2013 ; Revell et al, 2013 ; Klint et al, 2015 ; Murray et al, 2015 ; Murray et al, 2016 ; Shcherbatko et al, 2016 ; Rahnama et al, 2017 ; Zhang et al, 2018 ; Neff et al, 2020 ), the determinants of NaSpTx1 pharmacology at the potential pain target hNa V 1.3 ( Black et al, 1999 ; Kim et al, 2001 ; Hains et al, 2004 ; Hong et al, 2004 ; Garry et al, 2005 ; Lindia et al, 2005 ; Black et al, 2008 ; Chen et al, 2014 ; Tan et al, 2015 ; Xu et al, 2016 ) and epilepsy target hNa V 1.2 ( Menezes et al, 2020 ) have been largely ignored.…”

“…The readers are requested to follow Supplementary Table S1 for relative IC 50 values and Supplementary Figures S1 for dose-response curves. (C) Active residues in the surface of rSsp1a (PDB: 7SKC) Dongol et al, 2021 ), GpTx-1 (modeled on engineered GPTX-1, PDB: 6MK5) ( Murray et al, 2015 ), HwTx-IV (PDB: 2M4X) Revell et al, 2013 ) and m3-HwTx-IV (PDB: 5T3M) ( Wisedchaisri et al, 2021 ) were compared to identify which alanine mutation reduced the toxin potency at hNa V 1.7 as indicated by the respective color code (red or pink). All four toxins were aligned at NaSpTx1 signature motif WCK/R (W and K/R bolded).…”