Ligand determinants of fatty acid activation of the pronociceptive ion channel TRPA1

Background and purpose. Arachidonic acid (AA) and its derivatives are important modulators of cellular signalling. The transient receptor potential cation channel subfamily A, member 1 (TRPA1) is a cation channel with important functions in mediating cellular responses to noxious stimuli and inflammation. There is limited information about the interactions between AA itself and TRPA1, so we investigated the effects of AA and key ethanolamide and amino acid/neurotransmitter derivatives of AA on hTRPA1. Experimental approach. HEK 293 cells expressing hTRPA1 were studied by measuring changes in intracellular calcium ([Ca]i) with a fluorescent dye and by standard whole cell patch clamp recordings. Key results. AA (30 mu M) increased fluorescence in hTRPA1 expressing cells by 370% (notional EC50 13 mu M). The covalent TRPA1 agonist cinnamaldehyde (300 mu M) increased fluorescence by 430% (EC50, 11 mu M). Anandamide (230%) and N-arachidonoyl tyrosine (170%) substantially activated hTRPA1 at 30 mu M, however, N-arachidonoyl conjugates of glycine and taurine were less effective while N-acyl conjugates of 5-HT did not affect hTRPA1. Changing the acyl chain length or the number and position of double bonds reduced fatty acid efficacy at hTRPA1. Mutant hTRPA1 (Cys621, Cys641 and Cys665 changed to Ser) could be activated by AA (100 mu M, 40% of wild type) but not by cinnamaldehyde (300 mu M). Conclusions and implications. AA is a more potent activator of TRPA1 than its ethanolamide or amino acid/neurotransmitter derivatives and acts via a mechanism distinct from that of cinnamaldehyde, further underscoring the likelyhood of multiple pharmacologically exploitable sites on hTRPA1.