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

Given the important role of voltage-gated sodium (Na_V) channel-modulating spider toxins in elucidating the function, pharmacology, and mechanism of action of therapeutically relevant Na_V channels, we screened the venom from Australian theraphosid species against the human pain target hNa_V1.7. 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 hNa_V subtypes with a rank order of potency hNa_V1.7 > 1.6 > 1.2 > 1.3 > 1.1. rSsp1a inhibited hNa_V1.7, hNa_V1.2 and hNa_V1.3 without significantly altering the voltage-dependence of activation, inactivation, or delay in recovery from inactivation. However, rSsp1a demonstrated voltage-dependent inhibition at hNa_V1.7 and rSsp1a-bound hNa_V1.7 opened at extreme depolarizations, suggesting rSsp1a likely interacted with voltage-sensing domain II (VSD II) of hNa_V1.7 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.