Subpopulation of highly reactive cysteines underlie the activation of TRPA1 by electrophiles (1061.6)

Transient Receptor Potential Ankyrin 1 is a cation channel expressed on nociceptive sensory nerves. TRPA1 is activated by electrophiles following covalent modification (Michael Addition), and this mechanism contributes to nociceptor activation (and pain/defensive reflexes) during oxidative stress. However, the rate of electrophile‐induced TRPA1 currents (~1000 M‐1s‐1) is much faster than the electrophilic covalent modification of canonical Cys residues (<3 M‐1s‐1). The basis for this high reactivity is not known. Using overexpression of V5‐tagged TRP channels and pulse‐chase experiments with fluorescent electrophile BODIPY‐iodoacetamide (BD‐IA), we have studied TRP channel covalent modification following immunoprecipitation. Human TRPA1 is rapidly bound by BD‐IA (<60s, 10uM), with ~50% of the binding sensitive to a short pretreatment with IA. Other TRP channels are slowly bound by BD‐IA, but this binding is not blocked by IA pretreatment. As Michael Addition requires ionization of thiols we determined the pKa of TRP channel Cys. The IA‐blockable Cys residues of human TRPA1 had very low pKa (~3) compared to the IA‐insensitive Cys residues (>8). In addition, TRPA1 binding of electrophiles is not modified by intracellular polyphosphates (required for TRPA1 currents) or TRPA1 antagonists. We conclude that a subpopulation of reactive Cys is responsible for TRPA1’s unusual activation mechanism. Grant Funding Source : Supported by R01HL119802‐01