Lipid raft localization of the chemosensory cation channel TRPA1

TRPA1 is a calcium-permeable channel that functions as sensor for a wide variety of chemical irritants, including highly reactive electrophiles and redox agents. In addition, this channel is activated by a myriad of membrane-permeable compounds, through mechanisms that are poorly understood. One of the current hypotheses states that the activation of TRPA1 may be mediated by modifications of the lipid environment occurring due to the insertion of lipophilic compounds. However, so far not much is known about the localization of TRPA1 in the plasma membrane. In this study we gathered evidence for localization of this channel in lipid rafts. Using TIRF microscopy in HEK293T cells transfected with a mouse TRPA1 channel carrying a C-terminal mCherry tag we found high co-localization rate between this channel and the lipid raft maker cholera toxin. Ultracentrifugation of Triton-X insoluble fractions confirmed the co-expression of TRPA1 and the lipid raft marker flotillin-2 in the low density membrane fractions. Lipid raft disruption experiments further confirmed the cholesterol-related localization of TRPA1, with the major channel population found in higher density gradients. Modification of lipid rafts with the cholesterol-depleting agent M?-cyclodextrin or the sphingolipid hydrolase sphingomyelinase decreased the responses of known TRPA1 agonists such as bacterial lipopolysaccharides, thymol and allyl isothiocyanate. Taken together, these data indicate that TRPA1 is present in lipid rafts and that this location is required for normal channel activation by chemical agents.