1.2 TRPA1 expression pattern and membrane trafficking
Initially, it was believed that the TRPA1 channel expressed in the sensory neurons of dorsal root ganglia (DRG), trigeminal ganglia(TG) and nodose ganglia, is primarily involved in the nociception(Story et al. 2003; Bautista et al. 2005). However, growing evidences from different studies suggested non-neuronal expression of TRPA1 in various organs including the heart, lungs, brain, pancreas, gastrointestinal tract and urinary bladder etc. (De Logu et al., 2019; Kannler et al., 2018; Z. Wang et al., 2019). In the DRG, TRPA1 is highly expressed in small and medium-sized peptidergic afferent neurons (Nagata, Duggan et al., 2005; Patil et al., 2020), contrarily, there are reports which showed the expression of TRPA1 in non-peptidergic neurons (IB4 positive neurons) as well(Barabas, Kossyreva et al., 2012). TRPA1 expression on these sensory neurons is also associated with expression of neurotransmitters like CGRP and substance P, that are involved in nociception(Bautista et al., 2006; Peixoto-Neves, Soni et al., 2019). In the trigeminal ganglion (TG), unmyelinated and small myelinated neurons have TRPA1. TRPA1 expression is also detected in trigeminal sensory nuclei (TSN), the spinal dorsal horn (DH) and terminals of the superficial laminae of the trigeminal caudal nucleus (Vc)(Y. S. Kim et al., 2010; Zanotto, Merrill et al., 2007). In the spinal cord, substantia gelatinosa (SG) is a key site for receiving noxious inputs. Pre-synaptically located TRPA1 has been reported to transmit the noxious inputs by mediating glutamate release, hence, initiating a synaptic transmission onto the SG neurons(Inoue, Fujita et al., 2012). TRPA1 is also involved in the presynaptic glycinergic neurotransmission in the dorsal root horn(Cho, Jeong et al., 2012).
In the autonomic nervous system, TRPA1 expression has been reported in SCG (Sympathetic superior cervical ganglia) region(Smith, Beacham et al., 2004), although similar reports from other groups have failed to find the TRPA1 expression at RNA level in the SCG region(Nagata et al., 2005). TRPA1 has been associated with glutamate release in the brain stem(Sun, Bang et al., 2009; Yokoyama et al., 2011). Astrocytes, found throughout the brain, contribute to synapse formation and regulate neuronal functions. Since, TRPA1 is expressed in astrocytes, it has been speculated that TRPA1 can modulate neuronal functions too(Shigetomi, Jackson-Weaver et al., 2013). The presence of TRPA1 in the hippocampus has been reportedly linked with the activation of cannabinoid receptor(Koch et al., 2011). Enterochromaffin cells and myenteric nerves present in the small and large intestine also express TRPA1(Kong et al., 2016; Nozawa et al., 2009; T. et al., 2010). In the colon, pelvic neurons express TRPA1, axons of which originates from the DRG neurons at thoracolumbar and lumbosacral spinal levels(La, Schwartz et al., 2011). In the stomach, the pyloric region showed the expression of TRPA1 but not the cardia region as confirmed by quantitative Real-Time PCR andin-situ hybridization (Camacho et al., 2015). In the gastrointestinal tract, TRPA1 acts as a chemo-sensor for various stimuli from the luminal environment and modulates the function in the intestine along with intestinal odorant receptors(Kaji, Karaki et al., 2011). High TRPA1 expression has been detected in rat pancreatic beta islets(Cao et al., 2012). In addition to the neuronal expression of TRPA1 channel in various organs, non-neuronal expression of TRPA1 has been identified in lungs(Caceres et al., 2009; Nassini et al., 2012), mouse inner ear(Corey et al. 2004; Stepanyan et al. 2011), keratinocytes, fibroblasts(Jain et al.. 2011), enterochromaffin cells of human and rat colon (Doihara et al., 2009), melanocytes(Atoyan, Shander et al., 2009), and human dental pulp fibroblast(El Karim et al.. 2011)(figure 2 ).
The expression of TRPA1 on the cell membrane in different organs is modulated by several kinases and cellular regulators. Protein kinase A (PKA) and Phospholipase C (PLC) signaling play an important role in the maintenance of TRPA1 on the membrane(Bandell et al., 2004). Application of TRPA1 ligand mustard oil (AITC) and pharmacological activators of PKA/PLC signaling increased the expression of TRPA1 on the membrane by enhancing the SNARE (SNAP receptor) mediated vesicle fusion. Both stimuli have been reported to increase TRPA1 expression in-vitroin HEK293 cells. However, treatment with tetanus toxin attenuates the TRPA1 response to the second pulse of mustard oil in the cultured DRG neurons. Importantly, translocation or vesicular exocytosis of TRPA1 to the membrane depicts one of the mechanisms for increased expression of the protein during inflammation or when treated with agonists. Moreover, blockade of protein kinase A (PKA) and phospholipase C (PLC) signaling reduced mustard oil mediated increased expression of TRPA1 on the membrane(Schmidt et al. 2009). In an another experiment with non-electrophilic agonist carvacrol, TRPA1 expression was not found to be increased on the membrane, indicating the significance of the electrophilic nature of agonist in the trafficking of channel protein to the membrane(Meents, Fischer et al., 2016). Phosphorylation by Serine/Threonine kinases has been reported as an important modulator for subcellular targeting and gating of many TRP channels(Voolstra & Huber, 2014), but very little is known for this kind of modification in TRPA1 channel. The extensive large tandem ankyrin repeats present at the N-terminus of TRPA1 channel is a vital regulator of channel trafficking to the cell membrane, subcellular localization and homo-tetramerization of the channel protein. Cyclin dependent kinase 5 (Cdk5), is a member of the CDK (cyclin dependent kinase) family, primarily known for its role in mitosis(Cicero & Herrup, 2005), also regulates pain signaling pathways(Kumar Pareek, 2012; Utreras, Futatsugi et al., 2009). TRPA1 was found to be a substrate of Cdk5 phosphorylation, where Cdk5 modulates the channel activity or response to its agonists(Hall et al., 2018). Cdk5 has been investigated by Sulak et al ., for TRPA1 modulation in the DRG neurons and TRPA1 transfected HEK293 cells. They reported that serine 448 (S448) located in AR12 (Ankyrin repeat domain 12) domain of the TRPA1 channel acts as a target for direct phosphorylation by Cdk5 in the DRG sensory neurons. Cdk5 inhibition by roscovitine attenuated TRPA1 response to its agonists in DRG neurons but failed to abolish TRPA1 response to the agonists in transfected HEK 293 cells (Sulak, Ghosh et al., 2018). In a similar report, it has been reported that threonine 673 (T673), present outside the ankyrin (AR) domain in TRPA1, was the only possible site for phosphorylation by Cdk5(Hynkova, Marsakova et al., 2016). PKA, PLC, or Cdk5 mediated phosphorylation of the TRPA1 channel required a scaffolding A-kinase anchored protein (AKAP) which directly interacts with the TRPA1 to increase the basal phosphorylation of channel protein(Zimova et al., 2020).
Inflammation also induces trafficking of TRPA1 to the membrane. TNF-α increases the membrane expression of TRPA1. Inflammatory cytokines mediated trafficking is dependent on SNARE proteins including VAMP1 and Snap 25(Meng et al. 2016). Other than inflammatory signals and chemical agonists of the channel, temperature also modulates the TRPA1 expression. It has been found that both cold (below 4°C) and high (49°C) temperature stimulates TRPA1 expression(May et al. 2012).
The tumor suppressor gene CYLD affects the post-translational level of TRPA1 inside the cell. CYLD is a ubiquitin hydrolase enzyme that deubiquitinates the TRPA1 channel, hence increases the TRPA1 protein in the cell. However, oncogenic mutation of CYLD could affect the cellular expression of the TRPA1 protein(Stokes et al. 2006). Thus, ubiquitination pathways could be an important mechanism that regulates the cellular level of TRPA1. Another cellular regulator of TRPA1 is AMPK, the energy sensor of the cell. Activation of AMPK negatively regulates the expression of TRPA1 on the membrane. In DRG neurons, AMPK activity lowers after glucose treatment and simultaneously TRPA1 expression increases on the membrane(Wang et al.2018). Membrane components like cholesterol and lipid rafts also play an essential role in the maintenance and functionality of TRPA1 on the plasma membrane. Disruption of lipid rafts and depletion of cholesterol reduces the response of the channel towards AITC and its sensitivity to the chemical stimulation(Sághy et al., 2015). Recently, it has been found that TRPA1 localizes in the cholesterol rich domain in the plasma membrane as revealed by total internal fluorescence microscopy and density gradient centrifugation(Startek et al., 2019).Despite extensive reports on TRPA1 structure, its agonists-antagonists and physiological functions, molecular mechanisms underlying its intracellular regulation and maintenance are largely unknown. TRPA1 expression is modulated by both temperature and inflammatory cytokines but whether both use the same pathways is not clear. Similarly, membrane trafficking using SNARE dependent vesicular exocytosis has been exploited as a possible mechanism but ubiquitination pathways are still largely unknown. Further studies are required to get a clearer vision on channel modulation and trafficking.
TRPA1 Modulators
TRPA1 is activated by a variety of compounds including environmental irritants, pungent compounds, endogenous reactive mediators and pharmaceuticals (Bessac & Jordt, 2008). TRPA1 is a very attractive drug target for the development of analgesics and anti-inflammatory drugs, therefore pharmacology of this gating channel is of great importance. However, drug discovery efforts have been hampered due to the prominent differences between human and rodent TRPA1 homologues. It has been reported that many compounds which show agonistic effects with human TRPA1, can act as an antagonist in case of rodent TRPA1(Bianchi et al., 2012). A variety of compounds activate TRPA1 channel which can be broadly classified into two categories: electrophilic activators and non-electrophilic activators.
The first class of activators reacts with the thiol group of cysteine and lysine residue of the channel. With the help of mutagenesis, Hinmanet al ., identified the key cysteine residues involved in the electrophilic activation of the TRPA1 channel. These cysteine residues were C619, C639, and C663 (C621,C641 and C665 according to the human TRPA1 sequence in Uniprot ID: O75762) present between the last ankyrin repeat domain (ARD) and first transmembrane segment (S1) of the cytoplasmic N-terminal of the channel(Hinman, Chuang, Bautista, & Julius, 2006). In mice, cysteine’s C415, C422 and C622 (C414, C421 and C621 according to the human TRPA1 sequence in Uniprot ID: O75762), were identified by mass spectrometry and mutagenesis(Macpherson et al., 2007). Notably, mass spectrometric and cryo-electron microscopic studies have revealed C621 as an extraordinarily reactive site for electrophilic sensing in the TRPA1 channel(Bahia et al., 2016; Suo et al., 2020). Cysteine residues form the disulfide bridge when came in close proximity, potentially forming a ligand binding pocket. Mass spectrometric analysis of TRPA1 showed that upon binding with electrophilic agonists, there are conformational changes in critical cysteines (C193, C415, C463,C622, C634, and C666), along with some other cysteines (C31, C45, C66, C89,C105, C214, C259, C274, C541, C609, and C1087), hence support the above information that disulfide bonds are formed during binding between electrophilic activators and TRPA1 (Samanta et al., 2018). It has been reported that mutation of C633 and C651 which forms disulfide bonds lead to conformational changes in TRPA1 structure, making it non-responsive to electrophilic activators although there was a response to non-electrophilic activators(Babes et al., 2016).
Besides this continuously growing list of electrophilic activators of the TRPA1 channel, the other class of TRPA1 agonists comprises non-electrophilic compounds which modulate the channel without any covalent modifications. This class of activators has a wide range of compounds including natural compounds found in herbs like thymol (Lee et al., 2008), carvacrol (Xu, Delling et al., 2006), different classes of anesthetics(Leffler, Lattrell et al., 2011; Matta et al., 2008), nonsteroidal anti-inflammatory drugs (NSAIDs)(Hu et al., 2010) and different compounds used in therapeutics and cosmetics like parabens or alkyl esters of p-hydroxybenzoate(F. Fujita, Moriyama et al., 2007). Interestingly, some of the non-electrophilic modulators have bimodal action on TRPA1 activation and the best example of this is menthol (Karashima et al., 2007). Menthol which is a known TRPM8 agonist also activates mouse TRPA1 at low concentrations but inhibits the channel at higher concentrations(Karashima et al., 2007). Moreover it has been shown that menthol activates the human TRPA1, whereas non-mammalian TRPA1 does not respond to menthol, showing species differences of the channel(Xiao et al., 2008). Caffeine found in coffee represents another compound that showed species differences in the activation of TRPA1. Caffeine acts on amino acid Thr231 and Asp287 at the distal N-terminal region in mouse TRPA1 only, not in human TRPA1(Nagatomo & Kubo, 2008).
The list of various electrophilic and non-electrophilic compounds has been listed in Table 1.
The great importance of TRPA1 in pain, inflammation and many other diseases has initiated an increasing demand for the development of TRPA1 specific antagonists. The first TRPA1 antagonist was based on xanthine structure, symbolize as HC-030031 (Hydra Company), which was synthesized back in 2007. Glenmark company has also disclosed some antagonists based on phtalimide derivates and imidazo-uridine derivatives (for more detailed information on synthetic antagonists please see reviews by Talavera et al., 2019 and Nilius et al., 2012). These commercially available antagonists are non-electrophilic in nature and modulate TRPA1 channel activity by non-covalent modifications. Interestingly, there are electrophilic antagonists too, several oximes have been reported to have antagonistic activity towards TRPA1. Oxime AP-18 possesses both agonistic and antagonistic activity against the TRPA1 channel(DeFalco et al., 2010). Some natural compounds also have antagonistic properties for TRPA1. Eucalyptol (1,8-cineole) is an ether monoterpenoid present in eucalyptus oil that inhibits human TRPA1 currents stimulated by AITC, menthol and octanol(Takaishi et al., 2012). The menthol analogue 4-isopropylcyclohexanol inhibits hTRPA1(Takayama, Furue et al., 2017). Camphor (derived from Cinnamomum campharol) , cinnamaldehyde and nicotine, all have been shown to have both stimulating and inhibitory effects on TRPA1 depending upon the concentration(Alpizar et al., 2013; Lee et al., 2008; Talavera et al., 2009).