The local environment of cysteine 621 determines the rapid electrophilic adduction and activation of hTRPA1

Transient Receptor Potential Ankyrin 1 (TRPA1) is a non‐selective cation channel expressed largely on nociceptive sensory nerves. TRPA1 is rapidly activated by electrophiles, such as iodoacetamide, through covalent modification of intracellular cysteine (Cys) residues. We have previously shown that, at physiologically relevant exposures, iodoacetamide rapidly labels four Cys residues at positions 273, 621, 655, 1085. Of these, Cys residue at position 621 (C621) appears to be crucial for binding and activation of the human channel (hTRPA1). Interestingly, when comparing hTRPA1 with the electrophile‐insensitive rattlesnake homolog, while the amino acids lysine K620 and C621 are conserved the proline at P622 is not. We hypothesized that this rapid binding of C621 residue in human TRPA1 is mediated by neighboring amino acids K620 and P622 rendering it more sensitive to electrophilic adduction. We expressed V5‐His tagged hTRPA1 constructs in HEK293 cells including the wild‐type and the point mutants C621A, K620A and P622A. We used pulse‐chase experiments to label TRPA1 protein with iodoacetamide‐alkyne (IA‐alk), then used click chemistry to label with a fluorescent tag. Binding was measured using immunoprecipitated TRPA1 separated by SDS‐polyacrylamide gel electrophoresis and western blotting. TRPA1 channel activation was measured using ratiometric calcium imaging. The K620A mutant had the effect of greatly reducing electrophilic binding and activation of the channel. Modeling of deprotonation energies suggests that K620 contributes to C621 reactivity by reducing its p K a . The K620 mutant therefore likely reduces the probability that C621 will be thiolate and therefore able to rapidly react with electrophiles. The P622A mutant was also largely insensitive to electrophilic adduction and activation. IA‐alk binding was ~10% of that seen with the wild‐type channel. We hypothesize this is because, in the wild‐type hTRPA1, P622 induces a kink in the protein resulting in the positively charged K620 being closer to C621 compared with the nearby negatively charged glutamates, E625 and E681. The work described here highlights the importance of the amino acids K620 and P622 flanking C621. Our data demonstrate how the integrity of the local environment is essential to TRPA1 function as an electrophilic sensor. Support or Funding Information This work was supported by the National Heart Lung and Blood Institute in Bethesda, USA(R01‐HL119802‐S1) This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .