Identification of in vivo disulfide conformation of TRPA1 ion channel.
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.
Department of Pharmacology, School of Medicine, Case Western Reserve University,Cleveland, Ohio 44106, USA.
Liwen
L
Wang
Teresa L
TL
Cvetkov
Mark R
MR
Chance
Vera Y
VY
Moiseenkova-Bell
D000818:Animals; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry*; D015640:Ion Channel Gating / Q000502:physiology*; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000737:chemistry / Q000502:physiology; D051379:Mice; D009619:Nociceptors / Q000502:physiology; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae; D011984:Sensory Receptor Cells / Q000502:physiology; D013438:Sulfhydryl Compounds / Q000737:chemistry; D000074025:TRPA1 Cation Channel; D050051:Transient Receptor Potential Channels / Q000737:chemistry* / Q000502:physiology*
D004220:Disulfides; D008565:Membrane Proteins; D013438:Sulfhydryl Compounds; D000074025:TRPA1 Cation Channel; D050051:Transient Receptor Potential Channels; C475685:Trpa1 protein, mouse; D003545:Cysteine