title: A tarantula-venom peptide antagonizes the TRPA1 nociceptor ion channel by binding to the S1-S4 gating domain.
abstract: BACKGROUND
  The venoms of predators have been an excellent source of diverse highly specific peptides targeting ion channels. Here we describe the first known peptide antagonist of the nociceptor ion channel transient receptor potential ankyrin 1 (TRPA1).
  
  
  RESULTS
  We constructed a recombinant cDNA library encoding ∼100 diverse GPI-anchored peptide toxins (t-toxins) derived from spider venoms and screened this library by coexpression in Xenopus oocytes with TRPA1. This screen resulted in identification of protoxin-I (ProTx-I), a 35-residue peptide from the venom of the Peruvian green-velvet tarantula, Thrixopelma pruriens, as the first known high-affinity peptide TRPA1 antagonist. ProTx-I was previously identified as an antagonist of voltage-gated sodium (NaV) channels. We constructed a t-toxin library of ProTx-I alanine-scanning mutants and screened this library against NaV1.2 and TRPA1. This revealed distinct partially overlapping surfaces of ProTx-I by which it binds to these two ion channels. Importantly, this mutagenesis yielded two novel ProTx-I variants that are only active against either TRPA1or NaV1.2. By testing its activity against chimeric channels, we identified the extracellular loops of the TRPA1 S1-S4 gating domain as the ProTx-I binding site.
  
  
  CONCLUSIONS
  These studies establish our approach, which we term "toxineering," as a generally applicable method for isolation of novel ion channel modifiers and design of ion channel modifiers with altered specificity. They also suggest that ProTx-I will be a valuable pharmacological reagent for addressing biophysical mechanisms of TRPA1 gating and the physiology of TRPA1 function in nociceptors, as well as for potential clinical application in the context of pain and inflammation.
authors: Department of Cellular and Molecular Physiology, Yale School of Medicine, New Haven, CT 06520, USA; Department of Genetics, Yale School of Medicine, New Haven, CT 06520, USA; Program in Cellular Neuroscience, Neurodegeneration, and Repair, Yale School of Medicine, New Haven, CT 06520, USA.
authors: Junhong
authors: J
authors: Gui
authors: Department of Pharmacology, Yale School of Medicine, New Haven, CT 06520, USA.
authors: Boyi
authors: B
authors: Liu
authors: Department of Cellular and Molecular Physiology, Yale School of Medicine, New Haven, CT 06520, USA; Department of Genetics, Yale School of Medicine, New Haven, CT 06520, USA; Program in Cellular Neuroscience, Neurodegeneration, and Repair, Yale School of Medicine, New Haven, CT 06520, USA.
authors: Guan
authors: G
authors: Cao
authors: Department of Medicinal Chemistry and Molecular Pharmacology, College of Pharmacy, Purdue University, West Lafayette, IN 47907, USA.
authors: Andrew M
authors: AM
authors: Lipchik
authors: Department of Medicinal Chemistry and Molecular Pharmacology, College of Pharmacy, Purdue University, West Lafayette, IN 47907, USA.
authors: Minervo
authors: M
authors: Perez
authors: Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD 4072, Australia.
authors: Zoltan
authors: Z
authors: Dekan
authors: Centre for Advanced Imaging, The University of Queensland, Brisbane, QLD 4072, Australia.
authors: Mehdi
authors: M
authors: Mobli
authors: Centre for Biodiscovery and Molecular Development of Therapeutics, Queensland Tropical Health Alliance, James Cook University, Cairns, QLD 4870, Australia.
authors: Norelle L
authors: NL
authors: Daly
authors: Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD 4072, Australia.
authors: Paul F
authors: PF
authors: Alewood
authors: Department of Medicinal Chemistry and Molecular Pharmacology, College of Pharmacy, Purdue University, West Lafayette, IN 47907, USA.
authors: Laurie L
authors: LL
authors: Parker
authors: Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD 4072, Australia.
authors: Glenn F
authors: GF
authors: King
authors: Department of Cellular and Molecular Physiology, Yale School of Medicine, New Haven, CT 06520, USA.
authors: Yufeng
authors: Y
authors: Zhou
authors: Department of Pharmacology, Yale School of Medicine, New Haven, CT 06520, USA.
authors: Sven-Eric
authors: SE
authors: Jordt
authors: Department of Cellular and Molecular Physiology, Yale School of Medicine, New Haven, CT 06520, USA; Department of Genetics, Yale School of Medicine, New Haven, CT 06520, USA; Program in Cellular Neuroscience, Neurodegeneration, and Repair, Yale School of Medicine, New Haven, CT 06520, USA. Electronic address: michael.nitabach@yale.edu.
authors: Michael N
authors: MN
authors: Nitabach
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D004353:Drug Evaluation, Preclinical / Q000379:methods; D005260:Female; D015723:Gene Library; D006801:Humans; D008969:Molecular Sequence Data; D062551:NAV1.2 Voltage-Gated Sodium Channel / Q000378:metabolism; D009865:Oocytes; D010455:Peptides / Q000235:genetics / Q000494:pharmacology*; D017434:Protein Structure, Tertiary; D013111:Spider Venoms / Q000737:chemistry*; D050051:Transient Receptor Potential Channels / Q000037:antagonists & inhibitors*; D061567:Voltage-Gated Sodium Channel Blockers / Q000494:pharmacology; D029867:Xenopus Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism
chemical_list: D062551:NAV1.2 Voltage-Gated Sodium Channel; D010455:Peptides; D013111:Spider Venoms; D050051:Transient Receptor Potential Channels; D061567:Voltage-Gated Sodium Channel Blockers; D029867:Xenopus Proteins