Discrepancy between TRPA1 Activation by Reversible and Irreversible Electrophiles Suggests Involvement of Cytosolic Cofactors Other Than Polyphosphates

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 neurogenic inflammation in the peripheral nerve endings. TRPA1 can be 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, which leads 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, patch clamp studies have shown 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 NEM compared to AITC. Furthermore, single channel patch clamp experiments display reduced open probability with NEM compared to AITC and H2O2. 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. The data suggests that cytosolic factors other than polyphosphates are important for TRPA1 activation by irreversible electrophilic ligands.