Identifying Potential Agonists Of TRPA1 Using A Heterologous Expression System And Transgenic Mice

Abstract

Chemical irritants stimulate the trigeminal nerve through many different receptor proteins, including TRP channels. TRPA1, a highly conserved TRP channel, is known to be activated by over ninety compounds. As TRPA1 is promiscuous in nature, it was considered a likely receptor for stimuli activating the trigeminal nerve. In this study, thirteen stimuli were tested to see if they activated TRPA1. Of these stimuli, eight were potentially novel agonists of TRPA1. Using a fluorescent plate reader and the calcium-sensitive fluorescent dye FLUO-3AM, intracellular calcium levels of naive HEK and hTRPA1-HEK cells were monitored after exposure to a stimulus of interest. If a stimulus activated TRPA1, intracellular calcium levels increased resulting in increased fluorescence. Using the TRPA1 inhibitor HC-030031, elicitation of increases in intracellular calcium was confirmed to be due to TRPA1 activation for several compounds. Additionally, a behavioral aversion assay was conducted using wild type and TRPA1-/- mice to determine if TRPA1 was activated by the stimuli. Overall, five novel TRPA1 agonists (alpha-terpineol, amyl acetate, benzaldehyde, d-limonene, toluene) were identified. It remains unclear whether acetic acid, cyclohexanone and denatonium benzoate are TRPA1 agonists. Further study is necessary to determine the precise interactions of these compounds and TRPA1.