Abstract
The human Transient Receptor Potential A1 (hTRPA1) ion channel, also known as the wasabi receptor, acts as a biosensor of various potentially harmful stimuli. It is activated by a wide range of chemicals, including the electrophilic compound N-methylmaleimide (NMM), but the mechanism of activation is not fully understood. Here, we used mass spectrometry to map and quantify the covalent labeling in hTRPA1 at three different concentrations of NMM. A functional truncated version of hTRPA1 (Δ1-688 hTRPA1), lacking the large N-terminal ankyrin repeat domain (ARD), was also assessed in the same way. In the full length hTRPA1, the labeling of different cysteines ranged from nil up to 95% already at the lowest concentration of NMM, suggesting large differences in reactivity of the thiols. Most important, the labeling of some cysteine residues increased while others decreased with the concentration of NMM, both in the full length and the truncated protein. These findings indicate a conformational switch of the proteins, possibly associated with activation or desensitization of the ion channel. In addition, several lysines in the transmembrane domain and the proximal N-terminal region were labeled by NMM, raising the possibility that lysines are also key targets for electrophilic activation of hTRPA1.
Highlights
Animals have evolved a variety of biosensors to detect and avoid harmful conditions like detrimental chemicals and temperatures
In order to compare the labeling of cysteines and to probe for agonist-induced conformational changes of human Transient Receptor Potential A1 (hTRPA1), the solubilized proteins were individually incubated with three different molar ratios of NMM in respect to the total cysteines of the protein (Figure 1)
The NMM labeling demonstrated a concentration-dependent modification of certain cysteines in hTRPA1, Cys308 and Cys540 showed an increase in labeling whereas Cys651 and Cys834 showed decrease in labeling with higher concentrations of NMM
Summary
Animals have evolved a variety of biosensors to detect and avoid harmful conditions like detrimental chemicals and temperatures. The TRPA1 channel is activated by a large variety of chemicals, from exogenous plant-derived chemicals and environmental toxic reagents to endogenous inflammatory mediators, such as isothiocyanates (mustard and wasabi), cinnamaldehyde (cinnamon), allicin and diallyl disulphide (garlic), acrolein (smoke and chemotherapeutic metabolite) and nitroxyl [1,2]. To this date, more than 100 compounds including non-electrophilic compounds have been identified as TRPA1 activators and the number is still growing [1,2]. Studies have shown that such chemicals interact with TRPA1 by different mechanisms, e.g., covalent interaction of electrophilic compounds with free thiols of cysteine residues, non-covalent interaction and oxidation of cysteines that eventually promote the formation of disulphide bonds [4,5,6,7,8,9]
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