2.1 TRPA1 and Natural Activators
A vast number of pungent natural compounds activate TRPA1 channel. The most important activator of this class is AITC, responsible for fresh flavor of wasabi and pungency of mustard oil(Uchida, Miura et al., 2012). Other than AITC, wasabi also contains 6-(methyl sulfinyl)hexyl isothiocyanate (6-MSITC) and 6-(methylthio)hexyl isothiocyanate (6-MTITC), which act as an electrophilic activator of TRPA1(Uchida et al., 2012).
Another prominent agonist of TRPA1 is cinnamaldehyde, found in the oil of cinnamon (Cinnamonum verum) (Bandell et al., 2004). Cinnamaldehyde has been reported to causes a burning or tingling sensation and pain stimulation if taken orally, due to the activation of TRPA1(Alenmyr, Herrmann et al., 2011).
Garlic is known for centuries for its medicinal properties including anti-bacterial, anti-fungal, anti-cancer, anti-inflammatory effects etc.(Bayan, Koulivand et al., 2014; Hosseini & Hosseinzadeh, 2015). Allicin, an organo-sulfur pungent compound of garlic, is an electrophilic activator of TRPA1(Bautista et al. 2005). Allicin also acts as a precursor for its other derivatives like diallyl sulfide, diallyl disulfide and diallyl trisulfide during metabolism. Allicin and its derivatives also activates TRPV1 at lower potency as compared to TRPA1, but efficacy of TRPV1 activation is lower than that of capsaicin(Koizumi et al., 2009; Macpherson et al., 2005). Ajoene, an allicin derivative, does not activate the TRPA1 channel but enhances the activation by other electrophilic compounds including AITC and allicin. Diethyl disulfide, an allicin related compound, found in Durian fruit also activates TRPA1 channel to mediate the hyper thermic effects(Terada et al., 2014).
S-alkyl-S-alkenyl disulfides, a rare class of natural products found in the Ferula assa-foetida L . potently activates TRPA1 channel suggesting the role of TRPA1 in the potential beneficial health claims associated with the use of asafoetida as a spice(Shokoohinia et al., 2013).
Phenol compounds like carvacrol and propofol activates human TRPA1(Woll et al., 2017; Xu et al., 2006). The pungency of oregano has been attributed to the carvacrol mediated TRPA1 activation(Xu et al., 2006). It has been observed that intra-epidermal injections of TRPA1 agonist carvacrol to the humans cause dose dependent pain sensations which were reduced in the presence of TRPA1 antagonist A-967079(Schwarz, Namer et al., 2017). Thymol, a monoterpene phenol derivative of cymene is present in oil of the thyme plant, has bimodal action on the TRPA1 channel similar to the menthol. Activation of TRPA1 by thymol desensitize the channel for further exposure to AITC or thymol(Lee et al., 2008).Monoterpenoids such as p-cymene-3-carboxylic acid, 3-amino-p-cymene(Ortar et al., 2012) and limonene(Kaimoto et al., 2016) also activates the TRPA1 channel.
6-gingerol, a phenol derived compound, found in the essential oil of fresh ginger and cloves, activates TRPA1(YANG et al.. 2016). Eugenol, a phenylpropene present in cloves stimulates TRPA1(Chung et al., 2014). Oleocanthal is a natural phenolic compound, responsible for the pungency of extra virgin olive oil. It has been demonstrated that oleocanthal dependent stimulation of rodent trigeminal nervous system requires functional TRPA1(Peyrot Des Gachons et al., 2011). Piperine and its related compounds isopiperine, isochavicine, piperanine, piperolein A, piperolein B, and N-isobutyl-(2E,4E)-tetradeca-2,4-diamide are responsible for the spiciness of black pepper, also activate TRPA1(Okumura et al., 2010).
Ligustilide, found in Apium graveolens, Levisticum officinale, Angelica sinensis , Ligusticum chuanxiong , and North American traditional Medicine from Ligusticum portieri ), is a potent activator and moderate inhibitor of TRPA1 channel. Its bimodal action does not depend upon concentration, rather on its aromatization. For example, aromatization of ligustilide produces dehydroligustilide that has been reported to have inhibitory effects on TRPA1(Zhong et al., 2011).
Curcumin, a principle curcuminoid of turmeric, acutely activates and subsequently desensitize TRPA1 in HEK cells (human TRPA1 transfected cells) and mouse sensory neurons. Curcumin showed no effects on TRPV1 or TRPM8(Leamy, Shukla et al., 2011). TRPA1 is also activated by non-pungent compounds like capsiate, dihydrocapsiate and nordihydrocapsiate (all these capsinoids are found in sweet chili) to lower potency than TRPV1(Shintaku et al., 2012). Fatty acids of royal jelly activates TRPA1(Terada, Narukawa et al., 2011). Artepillin C contributes to the pungency of Brazilian green propolis, activates TRPA1 channel more potently than known activator AITC(Hata et al., 2012).
Plants, fungi and animals produce terpenes which act as protectors from predators and foragers. These terpenes have unsaturated aldehyde moieties that activates the TRPA1 channel. For example, sesquiterpene isovelleral, polygodial, miogadial and miogatrial activates TRPA1. α-, β-eudesmol and γ-eudesmol are non-electrophilic sesquiterpenes which also activates TRPA1 channel(Escalera, Von Hehn et al., 2008; Ohara et al., 2017; Terada et al., 2019). In a recent report by Terada et al . in 2019, human TRPA1 is found to be activated by terpenes. These terpenes were part of essential oil obtained from the byproduct of daidai juice processing. Out of the total 10 terpenes tested, they found that 5 of them (linalyl acetate, geranyl acetate, osthole, geranyl propionate, and neryl acetate) activate human TRPA1 but not TRPV1 or TRPM8. This study was done using transfected cell line which requires further validation(Terada et al.. 2019).
Recently, it has been demonstrated that three natural compounds cuminaldehyde (present in cumin), p-anisaldehyde (present in anise) and tiglic aldehyde (present in onion/garlic) from spice’s origin activate hTRPA1 in heterologous expression system and sensory neurons of DRG(Legrand, Merlini, de Senarclens-Bezençon, & Michlig, 2020).
Calcium ions dependent modulation
TRPA1 channel activity is modulated by a large number of endogenous activators including reactive oxygen species produced during inflammation, calcium ions, prostaglandins etc. Ca2+, one of the ubiquitous and most important regulators of the channel has been reported to have both potentiating (at low concentrations) and desensitizing (at high concentrations) effects. Initial studies on TRPA1 channel activity suggested the possible role of extracellular calcium in the amplification of TRPA1 response to mustard oil or Δ9-tetrahydrocannabinol (THC) in transfected HEK cells(Jordt et al., 2004). These effects were mediated by calcium entry through TRPA1 channel. Furthermore, these results were supported by Nagata et al ., they also speculated that extracellular calcium modulate the channel activity by binding to a site close to or within the channel(Nagata et al., 2005). TRPA1 channel modulation by Ca2+ is still a matter of debate. Extracellular calcium can also inactivate the channel; however, it has been shown that potentiation and inactivation of the TRPA1 viaCa2+ are two independent processes mediated by subsequent elevation in intracellular calcium ion concentrations(Y. Y. Wang, Chang, et al., 2008). The molecular mechanisms underlying Ca2+ dependent TRPA1 activation or inactivation are poorly understood. Initial reports demonstrated that Ca2+ ions bind to putative N-terminal EF hand motif of TRPA1 to activate the channel(Doerner, Gisselmann et al., 2007; Zurborg et al., 2007), but later studies showed that mutation in this region of TRPA1 does not affect the calcium dependent modulation of TRPA1(Nilius et al., 2011; Y. Y. Wang et al., 2008). An alternative hypothesis suggests that Ca2+ ions modulate TRPA1 channel activity by binding to acidic residues at the C-terminal. Deletion of 20 amino acids at C-terminal was reportedly does not affect the potentiation of the channel by Ca2+ ions or thiol-reactive groups, but reduces the Ca2+ dependent inactivation of the channel, thus supporting the potentiation-inactivation uncoupling mechanism. Simulations studies identified two amino acids (Asp1080 and Asp1082), critically important for the binding of Ca2+ ions at the C-terminal (Sura et al., 2012). Taken together, these studies indicate that the conserved acidic residues in the C-terminus of the TRPA1 channel significantly affect the channel modulation by Ca2+ ions. Some conserved residues between transmembrane domain 2 (TM2) and transmembrane domain 3 (TM3) have been identified with the help of structural analysis and electrophysiology. These residues were E788, Q791, Y799, N805 and E808, however E788 alone was found to be responsible for the major regulatory effects of calcium ions(Zhao, Lin King et al., 2020). Intriguingly, these calcium binding residues are highly conserved among TRP channels including TRPM2, TRPM4 and TRPM8(Autzen et al., 2018; Diver, Cheng et al., 2019; Y. Huang, Winkler et al., 2018; Z. Zhang, Tóth et al., 2018). Calmodulin (CaM), an intracellular calcium dependent protein has been recently reported to play an essential role in maintaining the TRPA1 channel activity in response to calcium ions concentrations. Notably, C-lobe of CaM directly interacts with the 17 amino acids long non-canonical calmodulin binding domain (CaMBD) present at the C-terminus of the TRPA1 channel. CaM acts as a calcium sensor and mutation in either Ca2+binding sites in CaM or CaM binding sites on TRPA1 renders the activation or desensitizing effects of calcium ions on TRPA1(Hasan, Leeson-Payne et al., 2017).
Role of TRPA1 agonism in prevention of diet induced Obesity
Obesity is a complex disease, resulted from the accumulation of excess fat in the body. There are many contributors to the development of obesity including endocrine disruptors, less sleep, cessation from smoking, less physical activity, various environmental and genetic factors. One of the important factors for the development of obesity is the imbalance in the energy expenditure which leads to the accumulation of fat in the different parts of the body. TRPA1 is majorly known for its role in pain sensation but growing evidence suggests its involvement in metabolic functions too. In this section, we have discussed the involvement of TRPA1 in regulating energy expenditure and thereby could be used as a target to combat weight gain, obesity and related complications.