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).