The Role of Lipid Rafts in the Localization and Function of the Chemosensory TRPA1 Channel

Abstract

Lipid rafts are highly specialized membrane domains characterized by high concentrations of cholesterol, sphingolipids and gangliosides. This distinctive composition results in lateral phase separation and generation of a liquid-ordered domains accommodating various receptors and channels involved in numerous cellular processes. Recently, several members of the Transient Receptor Potential channels superfamily (TRPCs, TRPM8, and TRPV1) have been shown to locate mainly in the lipid rafts and that this compartmentalization modulates their activation properties. Yet, not much is known about localization and interactions of TRPA1 with the surrounding plasma membrane. Since TRPA1 can be gated by a large number of electrophilic and non-electrophilic compounds, as well as by physiological stimuli such as cold we hypothesize that its localization in the lipid rafts could be a crucial factor influencing the channel activity. Exploiting microscopy and biochemical approaches, this study provides evidence for TRPA1 localization in lipid rafts. TIRF microscopy experiments in HEK293T cells transfected with a mouse or human TRPA1 channel carrying a C-terminal mCherry tag indicate high co-localization rate between channel and the lipid raft maker cholera toxin subunit B. Density gradient centrifugation of Triton-X insoluble fractions confirmed co-expression of TRPA1 and the lipid raft marker flotillin-2 in the low density membrane fractions. Lipid raft disruption experiments further affirmed the cholesterol-related localization of the channel, shifting its localization into higher density gradients. Modification of lipid rafts composition by cholesterol depletion (methyl-b-cyclodextrin) or sphingolipids hydrolysis (sphingomyelinase) decreased the responses of TRPA1 to lipopolysaccharides, thymol, allyl isothiocyanate and cold. Taken together, these results indicate that TRPA1 is present in lipid rafts and this location is essential for its normal activation by different agonists.