TRPA1 Can Be Activated By Acylation of Cysteine Residues

Laura Sadofsky, Andrew Boa, Alyn Morice. University of Hull, Hull, United Kingdom.

TRPA1 is widely believed to be a nociceptor. It is activated by a range of environmental irritants such as acrolein and toluene diisocyanate as well as plant products such as cinnamaldehyde and mustard oil. Recent publications have suggested that TRPA1 is activated by covalent modification of cysteine residues on the N terminus of the protein. Due to the diverse chemical structures of these activators the nature of the covalent bond with TRPA1 will naturally vary, but in the case of acrolein and cinnamaldehyde it is unclear whether this covalent link is via direct addition to the carbonyl group or via conjugate addition at the β-carbon. Using HEK293 cells permanently expressing cloned human TRPA1 (TRPA1-HEK) we performed calcium signalling experiments to determine the mechanism of TRPA1 activation with a series of related compounds which were capable of either direct and/or conjugate addition processes. Cinnamic acid, acrylic acid and acrylamide, three chemicals structurally related to acrolein/cinnamaldehyde, and which can react via conjugate addition, caused little or no TRPA1 activation up to 300μM. The N-hydroxy succinimide (NHS) ester of acrylic acid, however, caused a robust biphasic response in TRPA1 from 300nM to 100μM with a maximum response of 91% of calcium ionophore (maximum attainable response). We have so far tried a further six related compounds. Methyl vinyl ketone caused activation of TRPA1, from 300nM reaching a maximum at 100μM (49% of calcium ionophore). Mesityl oxide and methyl acrylate caused only small increases in intracellular calcium levels (15% of calcium ionophore at the maximum concentration tested, 1mM). The NHS ester of cinnamic acid resulted in activation of TRPA1 with a response similar to acrolein and a half log left shift compared to cinnamaldehyde (EC50 7.9μM compared to 44μM for cinnamaldehyde). The NHS ester of hydrocinnamic acid, which is incapable of reacting via a conjugate addition process, also activated TRPA1-HEK from 3μM with an EC50 of 30μM. All the TRPA1 agonists mentioned above failed to activate TRPV1-HEK or mock transfected HEK293 cells and acrylic acid NHS responses (3μM) could be inhibited following pre-incubation (10 minutes) of the cells with the TRPA1 specific antagonist HC-030031. The results suggest that activation of TRPA1 can also occur via an acylation of the cysteine residues.