Author ORCID Identifier
Document Type
Article
Publication Date
2-24-2012
Abstract
TRPA1 (transient receptor potential ankyrin 1) is an ion channel expressed in the termini of sensory neurons and is activated in response to a broad array of noxious exogenous and endogenous thiol-reactive compounds, making it a crucial player in chemical nociception. A number of conserved cysteine residues on the N-terminal domain of the channel have been identified as critical for sensing these electrophilic pungent chemicals, and our recent EM structure with modeled domains predicts that these cysteines form a ligand-binding pocket, allowing for the possibility of disulfide bonding between the cysteine residues. Here, we present a comprehensive mass spectrometry investigation of the in vivo disulfide bonding conformation and in vitro reactivity of 30 of the 31 cysteine residues in the TRPA1 ion channel. Four disulfide bonds were detected in the in vivo TRPA1 structure: Cys-666-Cys-622, Cys-666-Cys-463, Cys- 622-Cys-609, and Cys-666-Cys-193. All of the cysteines detected were reactive to N-methylmaleimide (NMM) in vitro, with varying degrees of labeling efficiency. Comparison of the ratio of the labeling efficiency at 300 μM versus 2 mM NMM identified a number of cysteine residues that were outliers from the mean labeling ratio, suggesting that protein conformation changes rendered these cysteines either more or less protected from labeling at the higher NMM concentrations. These results indicate that the activation mechanism of TRPA1 may involve N-terminal conformation changes and disulfide bonding between critical cysteine residues.
Keywords
disulfide, mass spectrometry, membrane proteins, protein conformation, TRP channels
Language
6176
Publication Title
Journal of Biological Chemistry
Grant
R01EB009688
Rights
© 2012 ASBMB. Currently published by Elsevier Inc; originally published by American Society for Biochemistry and Molecular Biology. This is an Open Access work distributed under the terms of the Creative Commons Attribution Licence (http://creativecommons.org/licenses/by/4.0/) You are free to copy, distribute and transmit the work, provided the original author and source are credited.
Creative Commons License
This work is licensed under a Creative Commons Attribution 4.0 International License.
Recommended Citation
Liwen Wang, Teresa L. Cvetkov, Mark R. Chance, Vera Y. Moiseenkova-Bell, Identification of in Vivo Disulfide Conformation of TRPA1 Ion Channel*, Journal of Biological Chemistry, Volume 287, Issue 9, 2012, Pages 6169-6176, ISSN 0021-9258, https://doi.org/10.1074/jbc.M111.329748..
Manuscript Version
Final Publisher Version