Absolute Modification of Highly Reactive Cysteines via N‐ethylmaleimide Evokes Full, Prolonged TRPA1 Activation

Abstract

A subgroup of sensory nerve fibers called nociceptors is stimulated by noxious stimuli and evokes defensive responses in organ systems. Transient Receptor Potential Ankyrin 1 (TRPA1) is a tetrameric, non‐selective, cationic channel that is expressed in sensory neurons that initiate afferent signaling in nociceptive fibers when activated. Activation of TRPA1 initiates nociceptive signaling to the central nervous system and elicits defensive behaviors and reflexes in organ systems and throughout the body. TRPA1 is activated by environmental pollutants, exogenous food chemicals and endogenous irritants that are created during inflammation and oxidative stress. TRPA1 agonists are electrophilic compounds that covalently modify highly reactive cysteines on the intracellular side of the channel. Our lab has identified four highly reactive cysteines (C273, C621, C665, C1085) that rapidly bind to electrophiles, with C621 having a greater reactivity than the other three cysteines. It is known that electrophilic binding of cysteines contributes to TRPA1 activation; however, the underlying mechanism of activation is poorly understood. In this study, we utilize cell‐attached single channel patch‐clamp to observe stochastic gating events of human TRPA1 (hTRPA1) channels that is transiently expressed in HEK293 cells to better understand the mechanism that leads to TRPA1 activation. Recordings of stochastic TRPA1 gating events during N‐ethylmaleimide (NEM) treatment reveal two different activation profiles: (1) a weak activation (low open probability [Po]) that persists over time; (2) a weak activation followed by a rapid, spontaneous increase to full activation (near 100% Po). Mean open time histogram analysis shows that the full TRPA1 activation profile has longer event open time compared to weak TRPA1 activation, which has mean open times that are comparable to open times of random events during baseline conditions. Also, the mean maximum open times during full TRPA1 activation is 75 times longer than weak TRPA1 activation mean maximum open times. Our results suggest that differences in the full or weak TRPA1 activation profiles are due to a difference in how the cysteines are modified via NEM. We hypothesize that full activation is the result of absolute C621 modification (each C621 residue on the four TRPA1 subunits) that fully opens the channel, whereas weak activation is the result of individual C621 modification events (not all four C621 residues) that do not have an additive effect on TRPA1 channel activation.