Electrophilic activation of the nociceptive channel TRPA1 requires a 6000‐fold increase in reactivity at cysteine 621 that is dependent on a neighboring lysine

Abstract

Mammalian TRPA1 is a cation channel expressed on nociceptive sensory nerves and contributes to pain/nociceptive responses to oxidative stress and inflammation. In particular TRPA1 is rapidly activated by oxidative stress‐associated electrophiles capable of covalent modification of cysteine residues. Putative ‘functionally electrophile‐sensitive’ Cys‐containing proteins must be put into context of antioxidant microenvironments containing mM glutathione and enzymes that accelerate electrophilic‐GSH reactions. Thus the mechanisms underlying the rapid reaction rates of TRPA1 Cys are fundamental to its ability to act as a competent sensor of oxidative stress and electrophilic damage. We have used physiologically relevant concentrations and exposures in both binding studies and functional experiments to offer mechanistic insights into the functional responses of TRPA1 to electrophiles. Human and mouse TRPA1 are rapidly covalently modified by electrophiles and this response is insensitive to TRPA1 inhibition or the absence of polyphosphates. Orthologs and paralogs insensitive to electrophiles display no rapid binding. Mass spectroscopy studies identified only 4 Cys rapidly modified: C273, C621, C665 and C1085. 98% of C621 were modified with 120s of 30uM iodoacetamide treatment. Only 3–4% of the other three Cys were modified. The second order reaction rate of C621 with iodoacetamide is >6000‐fold faster than typical Cys residues. The mutant C621A displays greatly reduced electrophilic binding and is not activated by iodoacetamide. Neighboring basic residues can increase thiol reactivity by promoting the thiolate state. Mutation of K620 eliminated electrophilic binding and electrophile‐induced activation, despite continued presence of C621. This data suggest that TRPA1 possesses a complex reactive Cys profile centering on the extraordinary reactivity of Cys 621, which is determined by its low pKa under the influence of its neighbor Lys 620. The reactivity of C621 is critical to TRPA1's functional sensitivity to oxidative stress. The extraordinary reactivity of TRPA1 C621 is likely fundamental to nociceptive responses to oxidative stress‐associated inflammation.Support or Funding InformationNHLBI R01HL119802 and R01HL119802‐S1