A Tarantula-Venom Peptide Antagonizes the TRPA1 Nociceptor Ion Channel by Binding to the S1–S4 Gating Domain

• Published in: Current biology 2014-03, Vol.24 (5), p.473-483
• Main Authors: Gui, Junhong, Liu, Boyi, Cao, Guan, Lipchik, Andrew M., Perez, Minervo, Dekan, Zoltan, Mobli, Mehdi, Daly, Norelle L., Alewood, Paul F., Parker, Laurie L., King, Glenn F., Zhou, Yufeng, Jordt, Sven-Eric, Nitabach, Michael N.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Binding Sites; Drug Evaluation, Preclinical - methods; Female; Gene Library; Humans; Molecular Sequence Data; NAV1.2 Voltage-Gated Sodium Channel - metabolism; Oocytes; Peptides - genetics; Peptides - pharmacology; Protein Structure, Tertiary; Spider Venoms - chemistry; Transient Receptor Potential Channels - antagonists & inhibitors; Voltage-Gated Sodium Channel Blockers - pharmacology; Xenopus Proteins - antagonists & inhibitors; Xenopus Proteins - chemistry; Xenopus Proteins - metabolism
• ISSN: 0960-9822; 1879-0445
• DOI: 10.1016/j.cub.2014.01.013

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). 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. 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. •The TRPA1 antagonist ProTx-I is identified by screening library of spider toxins•ProTx-I inhibits NaV channel by stabilizing closed state of voltage sensor•ProTx-I binds to TRPA1 and NaV by overlapping pharmacophore surfaces•The S1–S2 extracellular loop participates in ProTx-I binding to TRPA1 Gui et al. apply a new recombinant screening approach to the pharmacological diversity of spider venoms, thereby identifying ProTx-I—from the venom of the Peruvian green-velvet tarantula—as a potent peptide antagonist of human TRPA1 pain channel. ProTx-I inhibits TRPA1 most likely by binding to and stabilizing its closed state.