Mouse TRPA1 function and membrane localization are modulated by direct interactions with cholesterol.

Justyna B Startek,Debapriya Ghosh,Yeranddy A Alpizar,Bernd Nilius,Brett Boonen,Nele Van Ranst,Ariel Talavera,Thomas Voets,Alejandro López-Requena,Karel Talavera

doi:10.7554/elife.46084

Justyna B Startek, Debapriya Ghosh + Show 8 more

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https://doi.org/10.7554/elife.46084

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Abstract

The cation channel TRPA1 transduces a myriad of noxious chemical stimuli into nociceptor electrical excitation and neuropeptide release, leading to pain and neurogenic inflammation. Despite emergent evidence that TRPA1 is regulated by the membrane environment, it remains unknown whether this channel localizes in membrane microdomains or whether it interacts with cholesterol. Using total internal reflection fluorescence microscopy and density gradient centrifugation we found that mouse TRPA1 localizes preferably into cholesterol-rich domains and functional experiments revealed that cholesterol depletion decreases channel sensitivity to chemical agonists. Moreover, we identified two structural motifs in transmembrane segments 2 and 4 involved in mTRPA1-cholesterol interactions that are necessary for normal agonist sensitivity and plasma membrane localization. We discuss the impact of such interactions on TRPA1 gating mechanisms, regulation by the lipid environment, and role of this channel in sensory membrane microdomains, all of which helps to understand the puzzling pharmacology and pathophysiology of this channel.

Highlights

The detection of harmful stimuli such as noxious chemicals and changes in temperature and pressure are fundamental biological processes carried out by numerous cell types through diverse specialized receptors
We show that mouse TRPA1 is located preferably in cholesterol-rich domains and identify cholesterol recognition amino acid consensus (CRAC) motifs in the TM2 and TM4 segments that are implicated in the attenuation of chemical activation of mTRPA1 by cholesterol-depleting agents
With the use of total internal reflection fluorescence (TIRF) microscopy we found a population of highly mobile vesicular structures near the cellular membrane containing both mTRPA1-mCherry and the lipid raft marker cholera toxin B (Figure 1A and B, Video 1), as well as static areas where both fluorescent probes were colocalized at the membrane

Summary

Introduction

The detection of harmful stimuli such as noxious chemicals and changes in temperature and pressure are fundamental biological processes carried out by numerous cell types through diverse specialized receptors. TRPA1 has been implicated in sensory pathophysiology as detector of thermal (Story et al, 2003; Chen et al, 2013; Karashima et al, 2009; Hamada et al, 2008; Moparthi et al, 2016; Moparthi et al, 2014; Survery et al, 2016; Viswanath et al, 2003; Kwan et al, 2006; Vandewauw et al, 2018) and mechanical (Kwan et al, 2006; Kindt et al, 2007; Zhang et al, 2008; Kwan et al, 2009) stimuli This channel is best known for being activated by an extremely wide variety of noxious chemicals (Nilius and Appendino, 2013; Nilius et al, 2011; Zygmunt and Hogestatt, 2014; Startek et al, 2019b). This channel is activated by numerous non-electrophilic compounds, some of which have

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