Identification and characterization of novel proteins from Arizona bark scorpion venom that inhibit nav1.8, a voltage-gated sodium channel regulator of pain signaling

Tarek Mohamed Abd El-Aziz, Yucheng Xiao, Jake Kline, Harold Gridley, Alyse Heaston, Klaus D. Linse, Micaiah J. Ward, Darin R. Rokyta, James D. Stockand, Theodore R. Cummins, Luca Fornelli, Ashlee H. Rowe

Research output: Contribution to journalArticlepeer-review

Abstract

The voltage-gated sodium channel Nav1.8 is linked to neuropathic and inflammatory pain, highlighting the potential to serve as a drug target. However, the biophysical mechanisms that regulate Nav1.8 activation and inactivation gating are not completely understood. Progress has been hindered by a lack of biochemical tools for examining Nav1.8 gating mechanisms. Arizona bark scorpion (Centruroides sculpturatus) venom proteins inhibit Nav1.8 and block pain in grasshopper mice (Onychomys torridus). These proteins provide tools for examining Nav1.8 structure–activity relationships. To identify proteins that inhibit Nav1.8 activity, venom samples were fractioned using liquid chromatography (reversed-phase and ion exchange). A recombinant Nav1.8 clone expressed in ND7/23 cells was used to identify subfractions that inhibited Nav1.8 Na+ current. Mass-spectrometry-based bottom-up proteomic analyses identified unique peptides from inhibitory subfractions. A search of the peptides against the AZ bark scorpion venom gland transcriptome revealed four novel proteins between 40 and 60% conserved with venom proteins from scorpions in four genera (Centruroides, Parabuthus, Androctonus, and Tityus). Ranging from 63 to 82 amino acids, each primary structure includes eight cysteines and a “CXCE” motif, where X = an aromatic residue (tryptophan, tyrosine, or phenylalanine). Electrophysiology data demonstrated that the inhibitory effects of bioactive subfractions can be removed by hyperpolarizing the channels, suggesting that proteins may function as gating modifiers as opposed to pore blockers.

Original languageEnglish (US)
Article number501
JournalToxins
Volume13
Issue number7
DOIs
StatePublished - Jul 2021

Keywords

  • Bioactive proteins
  • Centruroides sculpturatus
  • Drug discovery
  • Hyperpolarization
  • Nav1.8
  • Neurotoxin
  • Onychomys torridus
  • Pain signaling
  • Scorpion venom
  • Voltage-gated sodium channel

ASJC Scopus subject areas

  • Toxicology
  • Health, Toxicology and Mutagenesis

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