A tarantula-venom peptide antagonizes the TRPA1 nociceptor Ion channel by binding to the S1-S4 gating domain

Junhong Gui, Boyi Liu, Guan Cao, Andrew M. Lipchik, Minervo Perez, Zoltan Dekan, Mehdi Mobli, Norelle L. Daly, Paul F. Alewood, Laurie L Parker, Glenn F. King, Yufeng Zhou, Sven Eric Jordt, Michael N. Nitabach

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SummaryBackground 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 Na V1.2. 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.

Original languageEnglish (US)
Pages (from-to)473-483
Number of pages11
JournalCurrent Biology
Issue number5
StatePublished - Mar 3 2014

Bibliographical note

Funding Information:
The authors thank Aiwei Sui for preparing the TRPA1 plasmid and mouse DRG neurons, Nathaniel Heintz for providing the GPI-tether construct, and L. Williamson for para and tipE clones. Work in the laboratory of M.N.N. is funded in part by NIH grants R01NS055035, R01NS056443, R21NS058330, and R01GM098931. Work in the laboratory of S.-E.J. is funded in part by NIH grants R01ES015056 and U01ES015674. G.F.K. acknowledges funding from the Australian Research Council (DP1095728).


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