Human Transient Receptor Potential Ankyrin 1 Research Articles

Structural analysis and theoretical studies of 2-(2-chlorobenzylidene) malononitrile (CS) “a riot controlling agent”

In solvent methanol, malononitrile and 2-chlorobenzaldehyde were stirred in the presence of base piperidine to synthesise the target chemical 2-(2-chlorobenzylidene) malononitrile (CS). The crystal structure of the target compound C10H5ClN2 has been elucidated through X-ray crystallographic analysis. It crystallises in the monoclinic crystal system within the P21 space group, containing four molecules per asymmetric unit. The unit cell dimensions are a = 3.8825(3) Å, b = 21.0202(18) Å, c = 21.3481(18) Å, and β = 95.096(4)°. The phenyl and malononitrile groups, which make up the molecular components, are each planar but slanted towards one another at a dihedral angle of 12.7° (4). Besides this, target molecule is also stabilized by hydrogen bonding, halogen bonding and π stacking interactions. Solid-state structure has been analysed through Hirshfeld surface analysis, including the evaluation of the different energy frameworks, indicating that the molecular sheets are primarily formed by hydrogen bonds and halogen bonds and the stabilization is dominated via the electrostatic energy contribution. Fingerprint plots were carried out to study the relative contribution of noncovalent interactions in the target compound CS. Further, the density functional theory calculations were employed using B3LYP hybrid functional with 6-311++G level basic set to optimize the structural coordinates in gas phase and in different solvents (chloroform, water, and methanol). The chemically active regions of the target compound were identified by the analysis of molecular electrostatic potential surface. Further, from PASS analysis and literature reports, molecular docking of title compounds was carried out with human transient receptor potential ankyrin 1 ion channel (hTRPA1) (PDB-ID 6PQO). ΔGb (docking energy) and inhibition constant was calculated to be −7.53 kcal/mol and 3.33 μM respectively, with the target ion channel.

Identification of the TRPA1 cannabinoid-binding site

Chronic pain accounts for nearly two-thirds of conditions eligible for medical cannabis licenses, yet the mechanisms underlying cannabis-induced analgesia remain poorly understood. The principal phytocannabinoids, the psychoactive Δ9-tetrahydrocannabinol (THC) and non-psychoactive cannabidiol (CBD), exhibit comparable efficacy in pain management. Notably, THC functions as an agonist of cannabinoid receptor 1 (CB1), whereas CBD shows minimal activity on CB1 and CB2 receptors. Elucidating the molecular targets through which phytocannabinoids modulate the pain system is required for advancing our understanding of the pain pathway and optimizing medical cannabis therapies. Transient receptor potential ankyrin 1 (TRPA1), a pivotal chemosensor in the pain pathway, has been identified as a phytocannabinoid target. Unlike most TRPA1 activators, phytocannabinoid activation is not mediated through the electrophilic binding site, suggesting an alternative mechanism. Here, we identified the human TRPA1 channel cannabinoid-binding site (CBS) and demonstrated that mutations at residue Y840 abolished responses to both THC and CBD at saturating concentrations, indicating a shared primary binding site. Molecular modeling revealed distinct interactions of THC and CBD with the Y840 residue within the CBS. Additionally, CBD binds to the adjacent general anesthetic binding site at oversaturating concentrations. Our findings define the CBS of TRPA1 as overlapping with and adjacent to binding sites for other allosteric activators, suggesting that TRPA1 possesses a highly adaptable domain for binding non-electrophilic activators. This underscores its unique role as a chemosensor in the pain pathway. Furthermore, our results provide new insights into the molecular mechanisms of cannabinoid-induced analgesia and identify novel targets for pain management therapies.

Critical amino acid residues regulating TRPA1 Zn2+ response: A comparative study across species

Cellular zinc ions (Zn2+) are crucial for signal transduction in various cell types. The transient receptor potential (TRP) ankyrin 1 (TRPA1) channel, known for its sensitivity to intracellular Zn2+ ([Zn2+]i), has been a subject of limited understanding regarding its molecular mechanism. Here, we employed metal ion-affinity prediction, three-dimensional structural modeling, and mutagenesis, utilizing data from the Protein Data Bank and AlphaFold Database, to elucidate the [Zn2+]i binding domain (IZD) structure composed by specific amino acid residues (AAs) in human (hTRPA1) and chicken TRPA1 (gTRPA1). External Zn2+ induced activation in hTRPA1, while not in gTRPA1. Moreover, external Zn2+ elevated [Zn2+]i specifically in hTRPA1. Notably, both hTRPA1 and gTRPA1 exhibited inherent sensitivity to [Zn2+]i, as evidenced by their activation upon internal Zn2+ application. The critical AAs within IZDs, specifically histidine at 983/984, lysine at 711/717, tyrosine at 714/720, and glutamate at 987/988 in IZD1, and H983/H984, tryptophan at 710/716, E854/E855, and glutamine at 979/980 in IZD2, were identified in hTRPA1/gTRPA1. Furthermore, mutations, such as the substitution of arginine at 919 (R919) to H919, abrogated the response to external Zn2+ in hTRPA1. Among single-nucleotide polymorphisms (SNP) at Y714 and a triple SNP at R919 in hTRPA1, we revealed that the Zn2+ responses were attenuated in mutants carrying the Y714 and R919 substitution to asparagine and proline, respectively. Overall, this study unveils the intrinsic sensitivity of hTRPA1 and gTRPA1 to [Zn2+]i mediated through IZDs. Furthermore, our findings suggest that specific SNP mutations can alter the responsiveness of hTRPA1 to extracellular and intracellular Zn2+.

Norterpene Cyclic Peroxides from the Marine Sponge Diacarnus spinipoculum, Inhibitors of Transient Receptor Potential Ankyrin 1.

Bioassay-guided isolation of the extract from the marine sponge Diacarnus spinipoculum showing inhibitory activity against human transient receptor potential ankyrin 1 (hTRPA1) resulted in the isolation of 12 norditerpene cyclic peroxides (1-12) and eight norsesterterpene cyclic peroxides (13-20). Among these, 10 (5-7, 11, 12, 16-20) are unprecedented analogs. Compounds with either a hydroxy (5, 11) or a methoxy (6, 12) group attached to the cyclohexanone moiety were obtained as epimeric mixtures at C-11, while compounds 4, 6, 10, and 12 are likely the artifacts of isolation. The absolute configurations of the new compounds were established based on an NMR-based empirical method and comparison of specific rotation values. Mosher ester analysis revealed the absolute configurations of compounds 17-20. The inhibitory activity of the isolated compounds against hTRPA1 varied significantly depending on their structures, with the norsesterterpenoid 19 displaying the most potent activity (IC50 2.0 μM).

Role of TRPA1 in Painful Cold Hypersensitivity.

Transient receptor potential ankyrin 1 (TRPA1) is a polymodal cation channel that plays a pivotal role in pain generation after exposure to irritant chemicals and is involved in the sensation of a wide variety of pathological pain. TRPA1 was first reported to be sensitive to noxious cold, but its intrinsic cold sensitivity still remains under debate. To address this issue, we focused on cold hypersensitivity induced by oxaliplatin, a platinum-based chemotherapeutic drug, as a peculiar adverse symptom of acute peripheral neuropathy. We and other groups have shown that oxaliplatin enhances TRPA1 sensitivity to its chemical agonists and reactive oxygen species (ROS). Our in vitro and animal model studies revealed that oxaliplatin, or its metabolite oxalate, inhibits hydroxylation of a proline residue within the N-terminus of human TRPA1 (hTRPA1) via inhibition of prolyl hydroxylase domain-containing protein (PHD), which induces TRPA1 sensitization to ROS. Although hTRPA1 is insensitive to cold, PHD inhibition endows hTRPA1 with cold sensitivity through sensing the small amount of ROS produced after exposure to cold. Hence, we propose that PHD inhibition can unveil the cold sensitivity of hTRPA1 by converting ROS signaling into cold sensitivity. Furthermore, in this review, we summarize the role of TRPA1 in painful cold hypersensitivity during peripheral vascular impairment.

Fluorescence Activity-Guided Isolation of Aaptamine Derivatives From the Marine Sponge Aaptos suberitoides and Their Inhibitory Activity Against Transient Receptor Potential Ankyrin 1

Objective: The aim of this research was to isolate and identify the natural products responsible for the biological and fluorescence activity of the extract from the marine sponge Aaptos suberitoides. Methods: Fluorescence activity-guided separation was performed after validation of the inhibitory activity of the extract against human transient receptor potential ankyrin 1 (hTRPA1) and subsequent measurement of its fluorescence activity. Three previously reported marine alkaloids, aaptamine, isoaaptamine, and 9- O-demethylaaptamine, were obtained and identified based on their nuclear magnetic resonance and high-resolution mass spectrometry data. Results: Aaptamine showed emission of the fluorescent light at a wavelength of 485 nm with a quantum yield of 31%. Isoaaptamine and 9- O-demethylaaptamine showed marginal fluorescence activity but promising inhibitory activity against hTRPA1 with IC50 values of 3.1 and 3.9 μM, respectively. Conclusions: This research demonstrates that natural fluorophores can be discovered in living organisms through photoactivity-guided isolation. Aaptamine, isolated as a result, provides an additional 1,6-naphthyridine fluorophore, while isoaaptamine and 9- O-demethylaaptamine suggest a chemical scaffold that could be used to study the function of ion channels.

Elucidation of the binding mode of organic polysulfides on the human TRPA1 receptor.

Introduction: Previous studies have established that endogenous inorganic polysulfides have significant biological actions activating the Transient Receptor Potential Ankyrin 1 (TRPA1) receptor. Organic polysulfides exert similar effects, but they are much more stable molecules, therefore these compounds are more suitable as drugs. In this study, we aimed to better understand the mechanism of action of organic polysulfides by identification of their binding site on the TRPA1 receptor. Methods: Polysulfides can readily interact with the thiol side chain of the cysteine residues of the protein. To investigate their role in the TRPA1 activation, we replaced several cysteine residues by alanine via site-directed mutagenesis. We searched for TRPA1 mutant variants with decreased or lost activating effect of the polysulfides, but with other functions remaining intact (such as the effects of non-electrophilic agonists and antagonists). The binding properties of the mutant receptors were analyzed by in silico molecular docking. Functional changes were tested by in vitro methods: calcium sensitive fluorescent flow cytometry, whole-cell patch-clamp and radioactive calcium-45 liquid scintillation counting. Results: The cysteines forming the conventional binding site of electrophilic agonists, namely C621, C641 and C665 also bind the organic polysulfides, with the key role of C621. However, only their combined mutation abolished completely the organic polysulfide-induced activation of the receptor. Discussion: Since previous papers provided evidence that organic polysulfides exert analgesic and anti-inflammatory actions in different in vivo animal models, we anticipate that the development of TRPA1-targeted, organic polysulfide-based drugs will be promoted by this identification of the binding site.

Activation of the human TRPA1 channel by different alkylating sulfur and nitrogen mustards and structurally related chemotherapeutic drugs

An important target in toxicology is the ion channel known as human transient receptor potential ankyrin 1 (hTRPA1). It is triggered by a variety of chemicals, including the alkylating chemical warfare agent sulfur mustard (SM). The activation potentials of structural analogs including O- and sesquimustard, nitrogen mustards (HN1, HN2, and HN3), and related chemotherapeutic drugs (bendamustine, cycylophosphamide, and ifosfamide) were examined in the current study. The aequorin assay was used to measure changes in intracellular calcium levels in human hTRPA1 overexpressing HEK293 cells. The XTT assay was used to determine cytotoxicity. The data presented here highlight that all investigated alkylating substances, with the exception of cyclophosphamide and ifosfamide, cause the activation of hTRPA1. Cytotoxicity and activation of hTRPA1 were found to be related. Compounds with high reactivity had higher cytotoxicity and vice versa. However, inhibiting hTRPA1 with the specific inhibitor AP18 could not reduce the cytotoxicity induced by alkylating agents. As a result, hTRPA1 does not play a significant role in the cytotoxicity of alkylating agents.

Canine, mouse, and human transient receptor potential ankyrin 1 (TRPA1) channels show different sensitivity to menthol or cold stimulation.

Transient receptor potential ankyrin 1 (TRPA1) is a nonselective cation channel that is activated by a variety of stimuli and acts as a nociceptor. Mouse and human TRPA1 exhibit different reactivity to some stimuli, including chemicals such as menthol as well as cold stimuli. The cold sensitivity of TRPA1 in mammalian species is controversial. Here, we analyzed the reactivity of heterologously expressed canine TRPA1 as well as the mouse and human orthologs to menthol or cold stimulation in Ca2+-imaging experiments. Canine and human TRPA1 exhibited a similar response to menthol, that is, activation in a concentration-dependent manner, even at the high concentration range in contrast to the mouse ortholog, which did not respond to high concentration of menthol. In addition, the response during the removal of menthol was different; mouse TRPA1-expressing cells exhibited a typical response with a rapid and clear increase in [Ca2+]i ("off-response"), whereas [Ca2+]i in human TRPA1-expressing cells was dramatically decreased by the washout of menthol and [Ca2+]i in canine TRPA1-expressing cells was slightly decreased. Finally, canine TRPA1 as well as mouse and human TRPA1 were activated by cold stimulation (below 19-20°C). The sensitivity to cold stimulation differed between these species, that is, human TRPA1 activated at higher temperatures compared with the canine and mouse orthologs. All of the above responses were suppressed by the selective TRPA1 inhibitor HC-030031. Because the concentration-dependency and "off-response" of menthol as well as the cold sensitivity were not uniform among these species, studies of canine TRPA1 might be useful for understanding the species-specific functional properties of mammalian TRPA1.

Species differences in activation of TRPA1 by resin additive-related chemicals relevant to indoor air quality.

Transient Receptor Potential Ankyrin 1 (TRPA1), which is expressed in the airways, has causative and exacerbating roles in respiratory diseases. TRPA1 is known as a target of sick building syndrome-related air pollutants, such as formaldehyde. Thus, an in vitro TRPA1 activation assay would be useful for predicting the potential risk of air pollution. In this study, we used human TRPA1 (hTRPA1)- and mouse TRPA1 (mTRPA1)-expressing cell lines to measure TRPA1 activation by the emerging indoor air pollutants 2-ethyl-1-hexanol (2-EH), a mixture of 2,2,4-trimethyl-1,3-pentanediol 1- and 3-monoisobutyrate (Texanol), and 2,2,4-trimethyl-1,3-pentanediol diisobutyrate (TXIB). The results indicated that 2-EH activated both hTRPA1 and mTRPA1 in a concentration-dependent manner, whereas TXIB did not activate hTRPA1 or mTRPA1. Texanol also activated hTRPA1 in a concentration-dependent manner. In contrast, a bell-shaped concentration-dependent curve was observed for mouse TRPA1 activation by Texanol, indicating inhibitory effects at a higher concentration range, which was also reported for menthol, a typical TRPA1 modulator. To further elucidate the mechanism underlying the species difference in TRPA1 activation by Texanol, V875G and G878V mutations were introduced into hTRPA1 and mTRPA1, respectively, which were reported to be key mutations for the inhibitory effect of menthol. These mutations switched the inhibitory effects of Texanol; thus, hTRPA1/V875G, but not mTRPA1/G878V, was inhibited at higher concentrations of Texanol. These results indicate that Texanol shares an interaction site with menthol. Overall, these findings suggest that careful interpretation is necessary when extrapolating rodent TRPA1-dependent toxicological effects to humans, especially with respect to the risk assessment of indoor air pollutants.

Species-Specific Activation of Transient Receptor Potential Ankyrin 1 by Phthalic Acid Monoesters.

Phthalic acid (PA) diesters are widely used in consumer products, as plasticizers, and are ubiquitous environmental pollutants. There is a growing concern about their adjuvant effect on allergic diseases. Although its precise mechanism remains unknown, possible involvement of transient receptor potential ankyrin 1 (TRPA1) has been suggested. Hence, in this study, the activation of human and mouse TRPA1s by a series of PA di- and monoesters was investigated using a heterologous expression system in vitro. Consequently, it was found that monoesters activated human TRPA1, where EC50 values were in the order of mono-hexyl > mono-heptyl > mono-n-octyl > mono-2-ethylhexyl > mono-isononyl and mono-isodecyl esters. Significant species differences in TRPA1 activation by PA monoesters were also discovered; PA monoesters activated human TRPA1 but not mouse TRPA1 in a concentration-dependent manner up to 50 µM. These findings suggest that PA esters may exert TRPA1-dependent adverse effects on humans, which have never been demonstrated in experimental animals.

TRPA1 modulation by Sigma-1 receptor prevents oxaliplatin-induced painful peripheral neuropathy.

Chemotherapy-induced peripheral neuropathy is a frequent, disabling side effect of anticancer drugs. Oxaliplatin, a platinum compound used in the treatment of advanced colorectal cancer, often leads to a form of chemotherapy-induced peripheral neuropathy characterized by mechanical and cold hypersensitivity. Current therapies for chemotherapy-induced peripheral neuropathy are ineffective, often leading to the cessation of treatment. Transient receptor potential ankyrin 1 (TRPA1) is a polymodal, non-selective cation-permeable channel expressed in nociceptors, activated by physical stimuli and cellular stress products. TRPA1 has been linked to the establishment of chemotherapy-induced peripheral neuropathy and other painful neuropathic conditions. Sigma-1 receptor is an endoplasmic reticulum chaperone known to modulate the function of many ion channels and receptors. Sigma-1 receptor antagonist, a highly selective antagonist of Sigma-1 receptor, has shown effectiveness in a phase II clinical trial for oxaliplatin chemotherapy-induced peripheral neuropathy. However, the mechanisms involved in the beneficial effects of Sigma-1 receptor antagonist are little understood. We combined biochemical and biophysical (i.e. intermolecular Förster resonance energy transfer) techniques to demonstrate the interaction between Sigma-1 receptor and human TRPA1. Pharmacological antagonism of Sigma-1R impaired the formation of this molecular complex and the trafficking of functional TRPA1 to the plasma membrane. Using patch-clamp electrophysiological recordings we found that antagonists of Sigma-1 receptor, including Sigma-1 receptor antagonist, exert a marked inhibition on plasma membrane expression and function of human TRPA1 channels. In TRPA1-expressing mouse sensory neurons, Sigma-1 receptor antagonists reduced inward currents and the firing of actions potentials in response to TRPA1 agonists. Finally, in a mouse experimental model of oxaliplatin neuropathy, systemic treatment with a Sigma-1 receptor antagonists prevented the development of painful symptoms by a mechanism involving TRPA1. In summary, the modulation of TRPA1 channels by Sigma-1 receptor antagonists suggests a new strategy for the prevention and treatment of chemotherapy-induced peripheral neuropathy and could inform the development of novel therapeutics for neuropathic pain.

Transient receptor potential ankyrin 1 channel: An evolutionarily tuned thermosensor.

The discovery of the role of the transient receptor potential ankyrin 1 (TRPA1) channel as a polymodal detector of cold and pain-producing stimuli almost two decades ago catalyzed the consequent identification of various vertebrate and invertebrate orthologues. In different species, the role of TRPA1 has been implicated in numerous physiological functions, indicating that the molecular structure of the channel exhibits evolutionary flexibility. Until very recently, information about the critical elements of the temperature-sensing molecular machinery of thermosensitive ion channels such as TRPA1 had lagged far behind information obtained from mutational and functional analysis. Current developments in single-particle cryo-electron microscopy are revealing precisely how the thermosensitive channels operate, how they might be targeted with drugs, and at which sites they can be critically regulated by membrane lipids. This means that it is now possible to resolve a huge number of very important pharmacological, biophysical and physiological questions in a way we have never had before. In this review, we aim at providing some of the recent knowledge on the molecular mechanisms underlying the temperature sensitivity of TRPA1. We also demonstrate how the search for differences in temperature and chemical sensitivity between human and mouse TRPA1 orthologues can be a useful approach to identifying important domains with a key role in channel activation.

Targeting novel human transient receptor potential ankyrin 1 splice variation with splice-switching antisense oligonucleotides.

Activation of transient receptor potential ankyrin 1 (TRPA1) channels by both environmental irritants and endogenous inflammatory mediators leads to excitation of the nerve endings, resulting in acute sensation of pain, itch, or chronic neurogenic inflammation. As such, TRPA1 channels are actively pursued as therapeutic targets for various pathological nociception and pain disorders. We uncovered that exon 27 of human TRPA1 (hTRPA1) could be alternatively spliced into hTRPA1_27A and hTRPA1_27B splice variants. The resulting channel variants displayed reduced expression, weakened affinity to interact with WT, and suffered from complete loss of function because of disruption of the C-terminal coiled-coil domain. Using a human minigene construct, we revealed that binding of splicing factor serine/arginine-rich splicing factor 1 (SRSF1) to the exonic splicing enhancer was critical for the inclusion of intact exon 27. Knockdown of SRSF1, mutation within exonic splicing enhancer, or masking SRSF1 binding with antisense oligonucleotides promoted alternative splicing within exon 27. Finally, antisense oligonucleotides-induced alternative splicing produced transcript and protein variants that could be functionally determined as diminished endogenous TRPA1 activity in human Schwann cell-line SNF96.2 and hiPSCs-derived sensory neurons. The outcome of the work could potentially offer a novel therapeutic strategy for treating pain by targeting alternative splicing of hTRPA1.

Sample preparation of the human TRPA1 ion channel for cryo-EM studies.

The transient receptor potential ankyrin 1 (TRPA1) ion channel is a member of the TRP channel family that is involved in sensing noxious stimuli that elicit pain and inflammation. Because of its critical physiological role and therapeutic importance, great efforts have been made to understand the structure and mechanism of TRPA1. Several human TRPA1 structures have been reported using single particle cryo-electron microscopy (cryo-EM) over the last 6 years. Here, we present a protocol for the heterologous expression, large-scale purification, and nanodisc reconstitution of the human TRPA1 channel for cryo-EM and biochemical studies.

A simple model for the assessment of the agonistic activity of dibenzazepine derivatives by molecular moieties

A simple approach is introduced to assess the agonistic activity of dibenzazepine derivatives including 11H-dibenz[b,e]azepine and the dibenz[b,f][1,4]oxazepine derivatives. Dibenzazepine derivatives show extremely potent activators of the human transient receptor potential ankyrin 1 receptor, which has a key role as a biological sensor. This new approach is based on molecular moieties for desk calculation of these compounds with more reliance on their answers as one could expect from available quantitative structure–activity relationship models. The measured agonistic activities of 31 diverse compounds in terms of pEC50 = −log (EC50), where EC50 is half-maximal effective concentration, are used as training (26 compounds) and test (5 compounds) sets. For the internal cross-validation, the calculated values of leave-one-out (Q2LOO) and the leave-more-out (Q2LMO) cross-validations are the same (0.99550) and very close to the coefficient of determination (R2 = 0.9965), which confirms that the new model is robust, well-behaved, and not over-fitted. The values of mean absolute percent error (MAPE) for training and test sets are 0.5943 and 0.8846, respectively, which are much lower than the outputs of two available complex comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA), i.e., the values of MAPE for training sets of CoMFA and CoMSIA are 0.8799 and 1.0412, while MAPE values for test sets of CoMFA and CoMSIA are 2.1591 and 1.7711, respectively.

Phospho-Mimetic Mutation at Ser602 Inactivates Human TRPA1 Channel.

The Transient Receptor Potential Ankyrin 1 (TRPA1) channel is an integrative molecular sensor for detecting environmental irritant compounds, endogenous proalgesic and inflammatory agents, pressure, and temperature. Different post-translational modifications participate in the discrimination of the essential functions of TRPA1 in its physiological environment, but the underlying structural bases are poorly understood. Here, we explored the role of the cytosolic N-terminal residue Ser602 located near a functionally important allosteric coupling domain as a potential target of phosphorylation. The phosphomimetic mutation S602D completely abrogated channel activation, whereas the phosphonull mutations S602G and S602N produced a fully functional channel. Using mutagenesis, electrophysiology, and molecular simulations, we investigated the possible structural impact of a modification (mutation or phosphorylation) of Ser602 and found that this residue represents an important regulatory site through which the intracellular signaling cascades may act to reversibly restrict or “dampen” the conformational space of the TRPA1 channel and promote its transitions to the closed state.

Transgenic Chlamydomonas Expressing Human Transient Receptor Potential Ankyrin 1 (TRPA1) Channels to Assess the Effect of Agonists and Antagonists.

Transient receptor potential ankyrin 1 (TRPA1) channel is an ion channel whose gating is controlled by agonists, such as allyl isothiocyanate (AITC), and temperature. Since TRPA1 is associated with various disease symptoms and chemotherapeutic side effects, it is a frequent target of drug development. To facilitate the screening of TRPA1 agonists and antagonists, this study aimed to develop a simple bioassay for TRPA1 activity. To this end, transgenic Chlamydomonas reinhardtii expressing human TRPA1 was constructed. The transformants exhibited positive phototaxis at high temperatures (≥20°C) but negative phototaxis at low temperatures (≤15°C); wild-type cells showed positive phototaxis at all temperatures examined. In the transgenic cells, negative phototaxis was inhibited by TRPA1 antagonists, such as HC030031, A-967079, and AP18, at low temperatures. Negative phototaxis was induced by TRPA1 agonists, such as icilin and AITC, at high temperatures. The effects of these agonists were blocked by TRPA1 antagonists. In wild-type cells, none of these substances had any effects on phototaxis. These results indicate that the action of TRPA1 agonists and antagonists can be readily assessed using the behavior of C. reinhardtii expressing human TRPA1 as an assessment tool.

Human TRPA1 is an inherently mechanosensitive bilayer-gated ion channel

The role of mammalian Transient Receptor Potential Ankyrin 1 (TRPA1) as a mechanosensor is controversial. Here, we report that purified human TRPA1 (hTRPA1) with and without its N-terminal ankyrin repeat domain responded with pressure-dependent single-channel current activity when reconstituted into artificial lipid bilayers. The hTRPA1 activity was abolished by the thiol reducing agent TCEP. Thus, depending on its redox state, hTRPA1 is an inherent mechanosensitive ion channel gated by force-from-lipids.

Bioelectronic Skin Based on Nociceptive Ion Channel for Human-Like Perception of Cold Pains.

A bioelectronic skin device based on nociceptive ion channels in nanovesicles is developed for the detection of chemical cold-pain stimuli and cold environments just like human somesthetic sensory systems. The human transient receptor potential ankyrin 1 (hTRPA1) is involved in transmission and modulation of cold-pain sensations. In the bioelectronic skin, the nanovesicles containing the hTRPA1 nociceptive ion channel protein reacts to cold-pain stimuli, and it is electrically monitored through carbon nanotube transistor devices based on floating electrodes. The bioelectronic skin devices sensitively detect chemical cold-pain stimuli like cinnamaldehyde at 10 fm, and selectively discriminate cinnamaldehyde among other chemical stimuli. Further, the bioelectronic skin is used to evaluate the effect of cold environments on the response of the hTRPA1, finding that the nociceptive ion channel responds more sensitively to cinnamaldehyde at lower temperatures than at higher temperatures. The bioelectronic skin device could be useful for a basic study on somesthetic systems such as cold-pain sensation, and should be used for versatile applications such as screening of foods and drugs.