Noxious compounds activate TRPA1 ion channels through covalent modification of cysteines

Abstract

The nervous system senses peripheral damage through nociceptive neurons that transmit a pain signal1,2. TRPA1 is a member of the Transient Receptor Potential (TRP) family of ion channels and is expressed in nociceptive neurons3,4,5. TRPA1 is activated by a variety of noxious stimuli, including cold temperatures, pungent natural compounds, and environmental irritants6,7,8,9,10,11. How such diverse stimuli activate TRPA1 is not known. We observed that most compounds known to activate TRPA1 are able to covalently bind cysteine residues. Here we use click chemistry to show that derivatives of two such compounds, mustard oil and cinnamaldehyde, covalently bind mouse TRPA1. Structurally unrelated cysteine-modifying agents such as iodoacetamide (IA) and (2-aminoethyl)methanethiosulphonate (MTSEA) also bind and activate TRPA1. We identified by mass spectrometry fourteen cytosolic TRPA1 cysteines labelled by IA, three of which are required for normal channel function. In excised patches, reactive compounds activated TRPA1 currents that were maintained at least 10 min after washout of the compound in calcium-free solutions. Finally, activation of TRPA1 by disulphide-bond-forming MTSEA is blocked by the reducing agent dithiothreitol (DTT). Collectively, our data indicate that covalent modification of reactive cysteines within TRPA1 can cause channel activation, rapidly signalling potential tissue damage through the pain pathway.

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Figure 1: TRPA1 is covalently modified by reactive compounds.
Figure 2: TRPA1 agonist binds to reactive cysteines, three of which are required for normal channel function.
Figure 3: Reactive compounds can cause sustained activation of TRPA1, and DTT reverses activation of TRPA1 by MTSEA.

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Acknowledgements

We thank T. Earley, M. Garrett, M. Petrus, J. Mathur, K. Spencer and the TSRI Mass Spectrometry Proteomics Core for technical help; and M. Bandell, A. Dhaka, J. Grandl, N. Hong, T. Jegla, L. Stowers and S. Trauger for valuable input. This research was supported by NIH grants and by Novartis Research Foundation. L.J.M. is the recipient of a Ruth Kirschstein Predoctoral Fellowship.

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Correspondence to Ardem Patapoutian.

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Macpherson, L., Dubin, A., Evans, M. et al. Noxious compounds activate TRPA1 ion channels through covalent modification of cysteines. Nature 445, 541–545 (2007). https://doi.org/10.1038/nature05544

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