**2. Activation mechanism of TRPA1 by reactive oxygen/nitrogen species and diverse chemical compounds**

TRPA1 is expressed in primary sensory neurons and mediates noxious pain sensations evoked by a wide range of reactive compounds. Electrophilic TRPA1 activators have environmental, dietary and endogenous origins and include acrolein (air pollutant), allyl isothiocyanate (in mustard), allicin and diallyl disulfide (in garlic), cinnamaldehyde (in cinnamon) and the proalgesic lipid peroxidation products 4-hydroxynonenal (4-HNE) and 15-deoxy-Δ12,14 prostaglandin J2 (15d-PGJ2 ) (**Figure 1A**) [30–33]. These compounds are collectively referred to as reactive carbonyl species (RCS). Many have highly reactive electrophilic carbon moieties such as an αβ-unsaturated carbonyl group. These moieties react with cysteine thiol groups in TRPA1, including those located between the N-terminal ARD and S1 (Cys621, Cys641, Cys665) of human TRPA1, which results in covalent modification by S-alkylation through a Michael addition that in turn activates TRPA1 (**Figure 1B**) [31]. In mouse, TRPA1, Cys415 and Cys422 in ARD and Cys622 between ARD and S1 are required for activation by reactive compounds [33]. In addition to cysteine residues, lysine (Lys708 in human TRPA1) is also reportedly involved in TRPA1 activation by reactive electrophilic species, although the contribution of this amino acid seems to be limited to the action of AITC [31].

TRPA1 is also activated by reactive oxygen/nitrogen species (ROS/RNS) and thus is regarded to be "redox-sensitive". TRPA1 is activated by many kinds of ROS and RNS, including hydrogen peroxide (H2 O2 ), hydroxyl radical (OH·), hypochlorite (OCl− ), nitric oxide (NO) and peroxynitrite (ONOO<sup>−</sup> ) [32, 34, 35] (**Figure 1A**). Oxidative stress endogenously generates lipid peroxidation products such as 4-NHE and 15d-PGJ2 , described above as RCS. However, ROS can activate TRPA1 independently of these peroxidation products, as evidenced by the finding that H<sup>2</sup> O2 -induced TRPA1 activation is reversed by the application of dithiothreitol

**Figure 1.** Chemical structure of TRPA1-activating reactive compounds (A) and reported target amino acid residues in TRPA1 (B).

(DTT), which reverses cysteine disulfide formation, nitrosylation and cysteine oxidation, but not Michael addition, without affecting the action of 4-NHE and 15d-PGJ<sup>2</sup> [32, 36]. This result suggests that ROS induce cysteine oxidation and disulfide formation between proximal cysteine residues in TRPA1.

TRPA1 activation by RNS is considered to be mediated by S-nitrosylation of cysteine residues, since TRPA1 activation by the NO donor SNAP was reversed by the application of DTT as described above [36]. Functional characterization of site-directed cysteine mutants of mouse TRPA1 demonstrated that modification of several cysteine residues (Cys621, Cys641, Cys665) involved in RCS activation mediates the action of ROS and RNS [34, 35]. However, mutation of cysteine and lysine residues could also affect channel activation by nonelectrophilic agonists, which would complicate the identification of amino acids responsible for covalent modification of TRPA1 [35]. TRPA1 activation by these reactive compounds leads to pain sensation that would diminish the likelihood of deleterious damages arising from adducts formed by these compounds and DNA or proteins that are associated with carcinogenesis and toxicity [37].
