How do AZ bark scorpion venom peptides inhibit Nav1.8 to block transmission of pain signals in grasshopper mice? How can this system provide insight into Nav1.8 gating mechanisms and improve efforts to develop non-addictive pain treatments?

Venom-derived peptide toxins from spiders, cone snails, sea anemones and scorpions bind voltage-gated sodium ion channels (Nav) disrupting channel activation and inactivation. Scorpion peptides have been used to dissect voltage-sensor structure and regulation of gating mechanisms. Nav1.8 has been linked to neuropathic and inflammatory pain, highlighting the potential to serve as a drug target. However, the biophysical mechanisms that regulate gating are not completely understood. While compounds have been identified that bind the pore to inhibit Na⁺ current, few toxin peptides are known that modify Nav1.8 gating. We discovered that AZ bark scorpion venom inhibits Nav1.8 expressed in the nociceptive neurons of predatory grasshopper mice. To identify the venom components that inhibit Nav1.8, we developed a venom-to-peptide pipeline for isolating and screening peptides with activity against Nav1.8. Our goal is to use these peptides as tools to study Nav1.8 gating properties. You can read all about our venom-to-peptide pipeline in Mohamed et al. 2021, Toxins.

Grasshopper mice have evolved amino acid substitutions in their Nav1.8 that impart binding sites to AZ bark scorpion venom peptides. When mice are stung by scorpions, the venom inhibits Nav1.8, decreasing compound action potentials in the dorsal root ganglia of nociceptive neurons. Ultimately, the venom blocks pain in these mice (Rowe et al. 2013, Science). Electrophysiological data suggest that the venom traps the Domain I voltage-sensor to stabilize Nav1.8 in a slow inactivated state (George et al. 2022, Frontiers in Pharmacology: Pharmacology of Ion Channels). Venom mining revealed multiple peptides that inhibit Nav1.8, ranging from a complete to a partial block of current. In order to characterize the inhibitory activity of venom peptides on Nav1.8 we are using an integrative approach:

• examine the effects of peptides on the electrophysiological properties of Nav1.8 gating
• map the binding sites between peptides and the channel
• build computational structural models of the peptide-Nav1.8 complex

A comparison of the effects of peptides on the electrophysiological properties of Nav1.8 from grasshopper mice with wildtype Nav1.8 from house mice will provide insight into the molecular and biophysical mechanisms underlying Nav1.8 gating. We are also combining chemical cross-linking mass spectrometry, site-directed mutagenesis and expression of recombinant peptide-encoding genes and Nav1.8 to map the amino acid residues in both peptides and channel that underlie structure-activity relationships. Structures of human Nav1.8 and scorpion peptide toxins provide templates for modeling grasshopper mice Nav1.8 bound to inhibitory peptides. Computational structural models of peptide-bound and unbound channels will increase our understanding of the structural basis for Nav1.8 gating. See our YouTube video below of AZ bark scorpion peptide NaTx36 bound to grasshopper mouse Nav1.8.

This study provides critical information on the basic biological activity of Nav1.8-inhibiting peptides. Moreover, we are discovering candidate peptides for developing high-throughput peptide expression – the first step toward using a site-directed mutagenesis and screening approach to identify peptides that inhibit human Nav1.8 without addiction.