title: The exceptionally high reactivity of Cys 621 is critical for electrophilic activation of the sensory nerve ion channel TRPA1.
abstract: Activation of the sensory nerve ion channel TRPA1 by electrophiles is the key mechanism that initiates nociceptive signaling, and leads to defensive reflexes and avoidance behaviors, during oxidative stress in mammals. TRPA1 is rapidly activated by subtoxic levels of electrophiles, but it is unclear how TRPA1 outcompetes cellular antioxidants that protect cytosolic proteins from electrophiles. Here, using physiologically relevant exposures, we demonstrate that electrophiles react with cysteine residues on mammalian TRPA1 at rates that exceed the reactivity of typical cysteines by 6,000-fold and that also exceed the reactivity of antioxidant enzymes. We show that TRPA1 possesses a complex reactive cysteine profile in which C621 is necessary for electrophile-induced binding and activation. Modeling of deprotonation energies suggests that K620 contributes to C621 reactivity and mutation of K620 alone greatly reduces the effect of electrophiles on TRPA1. Nevertheless, binding of electrophiles to C621 is not sufficient for activation, which also depends on the function of another reactive cysteine (C665). Together, our results demonstrate that TRPA1 acts as an effective electrophilic sensor because of the exceptionally high reactivity of C621.
authors.affiliation: Department of Molecular Pharmacology and Physiology, Morsani College of Medicine, University of South Florida, Tampa, FL 33612.
authors.forename: Parmvir K
authors.initials: PK
authors.lastname: Bahia
authors.affiliation: Department of Molecular Pharmacology and Physiology, Morsani College of Medicine, University of South Florida, Tampa, FL 33612.
authors.forename: Thomas A
authors.initials: TA
authors.lastname: Parks
authors.affiliation: Department of Molecular Pharmacology and Physiology, Morsani College of Medicine, University of South Florida, Tampa, FL 33612.
authors.forename: Katherine R
authors.initials: KR
authors.lastname: Stanford
authors.affiliation: Department of Molecular Medicine, Morsani College of Medicine, University of South Florida, Tampa, FL 33612.
authors.forename: David A
authors.initials: DA
authors.lastname: Mitchell
authors.affiliation: Department of Cell Biology, Microbiology, and Molecular Biology, College of Arts and Sciences, University of South Florida, Tampa, FL 33620.
authors.forename: Sameer
authors.initials: S
authors.lastname: Varma
authors.affiliation: Department of Cell Biology, Microbiology, and Molecular Biology, College of Arts and Sciences, University of South Florida, Tampa, FL 33620.
authors.forename: Stanley M
authors.initials: SM
authors.lastname: Stevens
authors.affiliation: Department of Molecular Pharmacology and Physiology, Morsani College of Medicine, University of South Florida, Tampa, FL 33612 ttaylorc@health.usf.edu.
authors.forename: Thomas E
authors.initials: TE
authors.lastname: Taylor-Clark
mesh_terms: D019943:Amino Acid Substitution; D001665:Binding Sites; D015220:Calcium Channels / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003545:Cysteine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D015640:Ion Channel Gating; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D055672:Static Electricity; D000074025:TRPA1 Cation Channel; D050051:Transient Receptor Potential Channels / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D015220:Calcium Channels; D009419:Nerve Tissue Proteins; D000074025:TRPA1 Cation Channel; C473350:TRPA1 protein, human; D050051:Transient Receptor Potential Channels; D003545:Cysteine