Discrepancy between TRPA1 activation by reversible and irreversible electrophiles suggests involvement of cytosolic cofactors other than polyphosphates.

Abstract

Transient Receptor Potential Ankyrin 1 (TRPA1) is a non‐selective cation channel that is expressed in a subset of nociceptive afferent sensory nerves. When activated, TRPA1 can evoke nociceptive responses (e.g. pain and defensive reflexes), as well as peripheral neurogenic inflammation. TRPA1 is activated by a host of endogenous (inflammatory mediators) and exogenous (pollutants, food) agonists. Many TRPA1 agonists are electrophilic compounds that covalently modify reactive cysteine residues on TRPA1, leading to activation of the channel. Electrophiles can bind through reversible or irreversible reactions based on the structure of the compound. Here, patch clamp techniques and calcium imaging have been used to characterize human TRPA1 (hTRPA1) channel activity (expressed in HEK293 cells) in the presence of reversible and irreversible electrophiles. Calcium imaging shows that both reversible electrophiles (e.g. allylisothiocyanate [AITC]) and irreversible electrophiles (e.g. iodoacetamide [IA] and N‐ethylmaleimide [NEM]) rapidly induce TRPA1 activation. In contrast, our patch clamp studies show a discrepancy between irreversible and reversible electrophilic TRPA1 activation. Whole cell patch clamp studies exhibit a reduction in the kinetics and magnitude of TRPA1 activation by IA and NEM compared to AITC. Furthermore, single channel patch clamp experiments display reduced open probability with IA and NEM compared to AITC. Both whole cell patch and excised patch clamp recordings were performed in the presence of 5mM polyphosphates. In preliminary gramicidin perforated patch recordings there was no difference in NEM and AITC‐evoked TRPA1 activation. Thus in intact cellular assays (Ca2+ imaging and gramicidin‐perforated patch) there is little difference in TRPA1 activation by reversible and irreversible electrophiles, whereas in dialyzed cellular assays (whole cell patch and excised patch) irreversible electrophiles evoked reduced TRPA1 activation compared to reversible electrophiles. The data suggests that cytosolic factors other than polyphosphates are important for TRPA1 activation by irreversible electrophilic ligands.Support or Funding InformationNHLBI R01 (HL119802) and Supplement (HL119802‐S1)