Mechanisms of peptide-mediated inhibition of Nav1.8 that block pain signals
How do specific toxin peptides in venom inhibit Nav1.8 to block transmission of pain signals in scorpion mice? How can this system provide insight into Nav1.8 gating mechanisms and improve efforts to develop non-addictive pain treatments?
Venom-derived toxins (peptides) from spiders, cone snails, sea anemones and scorpions bind voltage-gated sodium ion channels (VGSC) disrupting channel activation and inactivation. In particular, 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 toxins are known that modify Nav1.8 gating. We discovered that AZ bark scorpion venom inhibits Nav1.8 expressed in the nociceptive neurons of predatory scorpion mice. To identify the venom components that inhibit Nav1.8, we developed a venom-to-peptide pipeline for isolating peptides and screening them for activity against Nav1.8. Our goal is to use these peptides as tools to study Nav1.8 gating properties.
Scorpion 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. Electrophysiological data suggest that the venom traps the Domain I voltage-sensor to stabilize Nav1.8 in a slow inactivated state. Venom mining revealed five 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 models of the peptide-Nav1.8 complex
A comparison of the effects of peptides on the electrophysiological properties of Nav1.8 from scorpion 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. A computational model of the human Nav1.8 (SWISS-MODEL repository) and crystal structures of scorpion toxin peptides (Protein Data Bank) provide templates for modeling Nav1.8 bound to inhibitory peptides. Computational models of peptide-bound and unbound channels will increase our understanding of the structural basis for Nav1.8 gating.
This study will provide critical information on the basic biological activity of Nav1.8-inhibiting peptides. Moreover, the project will yield candidate peptides necessary for developing high-throughput peptide synthesis – the first step toward using an amino acid mutagenesis and screening approach to identify peptides that would inhibit human Nav1.8 without addiction.
Venom to Peptide Pipeline
Step 1: Scorpions are collected at night using blacklight. The crude venom is extracted and pooled from multiple specimens.
Step 2: Reverse Phase high performance liquid chromatography (HPLC) is used to fraction venom. Each peak may contain one or more peptides.
Step 3: Venom fractions from step 2 are tested on grasshopper mice Nav1.8 (OtNav1.8) expressed in ND7/23 cells to identify which region of the venom inhibits OtNav1.8.
Step 4: Ion Exchange Chromatograpy (IEC) is used to subfraction the region of the venom with the strongest inhibitory activity against OTNav1.8 into individual peptides. Individual peptides are collected for further testing of electrophysiological effects on OtNav1.8.
Step 5: Hybrid Mass Spectroscopy (M S), a combination of “top down” and “bottom up” M S, is used to determine the mass and partial amino acid sequence of peptides.
Step 6: Complete sequence for peptides is obtained by matching the mass and partial amino acid sequence from step 5 with sequences from the C. sculpturatus venom transcriptome (transcriptome provided by Darin Rokyta, FSU).
Step 7: The genes for individual peptides that inhibit OtNav1.8 are inserted into the pET-22b vector for expression in BL21 cells (University of Oklahoma Protein Production Core). Peptide expression vectors provide a template for downstream site-directed mutagenesis to identify amino acids that are critical for binding to OtNav1.8