Complete NEM Modification of Highly Reactive Cysteines Induces Full TRPA1 Activation

Abstract

Transient Receptor Potential Ankyrin 1 (TRPA1) is a tetrameric, non‐selective, cationic channel that is expressed in the dorsal root ganglia, trigeminal ganglia, and vagal ganglia and is activated by harmful chemicals including pollutants and endogenous irritants produced during inflammation and oxidative stress. Many irritants activate TRPA1 via electrophilic modification of highly reactive cysteine residues located on the cytosolic side of the channel, however the underlying processes that precede activation are poorly understood. Our lab has identified four highly reactive cysteines on TRPA1 (thus 16 highly reactive cysteines per channel) that rapidly bind electrophiles: C273, C621, C665 and C1085. C621 was the most reactive and point mutation studies show that both C621 and C665 are required for activation. In this study, we use cell‐attached single‐channel patch clamp analyses of transiently expressed human TRPA1 (hTRPA1) in HEK293 cells to determine the relationship between irreversible electrophilic binding and channel activity. We recorded stochastic gating events of single channels in response to N‐ethylmaleimide (NEM) treatment. Our open probability (Po) analysis indicates that NEM evoke two distinct hTRPA1 activation profiles: (1) weak activation (low Po) throughout NEM treatment which does not increase with time; (2) weak activation which then suddenly and spontaneously progresses to full activation (near 100% Po). Open time histogram analyses suggest that the weak activation state evoked by NEM has similar open times to spontaneous hTRPA1 activation in control conditions; whereas the full activation state has significantly longer open times. We conclude that the weak and full hTRPA1 activation states by NEM are due to differences in progressive cysteine modification. Furthermore, we suggest that full activation is not the summation of multiple weak states, but is instead a separate activation state that can only be evoked by a particular complement of cysteine modifications.Support or Funding InformationThis research is funded by the National Heart, Lung and Blood Institute (5R01HL119802‐S1).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.