Human Transient Receptor Potential Ankyrin 1 Channel: Structure, Function, and Physiology.

The transient receptor potential ion channel TRPA1 is a Ca2+-permeable nonselective cation channel widely expressed in sensory neurons, but also in many nonneuronal tissues typically possessing barrier functions, such as the skin, joint synoviocytes, cornea, and the respiratory and intestinal tracts. Here, the primary role of TRPA1 is to detect potential danger stimuli that may threaten the tissue homeostasis and the health of the organism. The ability to directly recognize signals of different modalities, including chemical irritants, extreme temperatures, or osmotic changes resides in the characteristic properties of the ion channel protein complex. Recent advances in cryo-electron microscopy have provided an important framework for understanding the molecular basis of TRPA1 function and have suggested novel directions in the search for its pharmacological regulation. This chapter summarizes the current knowledge of human TRPA1 from a structural and functional perspective and discusses the complex allosteric mechanisms of activation and modulation that play important roles under physiological or pathophysiological conditions. In this context, major challenges for future research on TRPA1 are outlined.

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 used 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 AAs residues 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 (SNPs) 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+.

Single amino acids set apparent temperature thresholds for heat-evoked activation of mosquito transient receptor potential channel TRPA1.

Animals detect heat using thermosensitive transient receptor potential (TRP) channels. In insects, these include TRP ankyrin 1 (TRPA1), which in mosquitoes is crucial for noxious heat avoidance and thus is an appealing pest control target. However, the molecular basis for heat-evoked activation has not been fully elucidated, impeding both studies of the molecular evolution of temperature sensitivity and rational design of inhibitors. In TRPA1 and other thermosensitive TRPs, the N-terminal cytoplasmic ankyrin repeat (AR) domain has been suggested to participate in heat-evoked activation, but the lack of a structure containing the full AR domain has hindered our mechanistic understanding of its role. Here, we focused on elucidating the structural basis of apparent temperature threshold determination by taking advantage of two closely related mosquito TRPA1s from Aedes aegypti and Culex pipiens pallens with 86.9% protein sequence identity but a 10 °C difference in apparent temperature threshold. We identified two positions in the N-terminal cytoplasmic AR domain of these proteins, E417 (A. aegypti)/Q414 (C. pipiens) and R459 (A. aegypti)/Q456 (C. pipiens), at which a single exchange of amino acid identity was sufficient to change apparent thresholds by 5 to 7 °C. We further found that the role of these positions is conserved in TRPA1 of a third related species, Anopheles stephensi. Our results suggest a structural basis for temperature threshold determination as well as for the evolutionary adaptation of mosquito TRPA1 to the wide range of climates inhabited by mosquitoes.

Selective activation of TRPA1 ion channels by nitrobenzene skin sensitizers DNFB and DNCB.

2, 4-dinitrofluorobenzene (DNFB) and 2, 4-dinitrochlorobenzene (DNCB) are well known as skin sensitizers that can cause dermatitis. DNFB has shown to more potently sensitize skin; however, how DNFB and DNCB cause skin inflammation at a molecular level and why this difference in their sensitization ability is observed remain unknown. In this study, we aimed to identify the molecular targets and mechanisms on which DNFB and DNCB act. We used a fluorescent calcium imaging plate reader in an initial screening assay before patch-clamp recordings for validation. Molecular docking in combination with site-directed mutagenesis was then carried out to investigate DNFB and DNCB binding sites in the TRPA1 ion channel that may be selectively activated by these tow sensitizers. We found that DNFB and DNCB selectively activated TRPA1 channel with EC50 values of 2.3 ± 0.7 µM and 42.4 ± 20.9 µM, respectively. Single-channel recordings revealed that DNFB and DNCB increase the probability of channel opening and act on three residues (C621, E625, and Y658) critical for TRPA1 activation. Our findings may not only help explain the molecular mechanism underlying the dermatitis and pruritus caused by chemicals such as DNFB and DNCB, but also provide a molecular tool 7.5-fold more potent than the current TRPA1 activator allyl isothiocyanate (AITC) used for investigating TRPA1 channel pharmacology and pathology.

Human TRPV1 and TRPA1 are receptors for bacterial quorum sensing molecules.

In this study, we investigated the activation of TRPV1 and TRPA1 by N-acyl homoserine lactones, quorum sensing molecules produced by Gram-negative bacteria, and the inhibitory effect of TRPV1 and TRPA1 by autoinducing peptides (AIPs), quorum sensing molecules produced by Gram-positive bacteria, using human embryonic kidney 293T cell lines stably expressing human TRPV1 and TRPA1, respectively. As a result, we found that some N-acyl homoserine lactones, such as N-octanoyl-L-homoserine lactone (C8-HSL), N-nonanoyl-L-homoserine lactone (C9-HSL) and N-decanoyl-L-homoserine lactone (C10-HSL), activated both TRPV1 and TRPA1. In addition, we clarified that some N-acyl homoserine lactones, such as N-3-oxo-dodecanoyl-L-homoserine lactone (3-oxo-C12-HSL), only activated TRPV1 and N-acyl homoserine lactones having saturated short acyl chain, such as N-acetyl-L-homoserine lactone (C2-HSL) and N-butyryl-L-homoserine lactone (C4-HSL), only activated TRPA1. Furthermore, we found that an AIP, simple linear peptide CHWPR, inhibited both TRPV1 and TRPA1 and peptide having thiolactone ring DICNAYF, the thiolactone ring were formed between C3 to F7, strongly inhibited only the TRPV1. Although the specificity of TRPV1 and TRPA1 for quorum sensing molecules was different, these data suggest that both TRPV1 and TRPA1 would function as receptors for quorum sensing molecule produced by bacteria. Graphical Abstract.

TRPA1: Pharmacology, natural activators and role in obesity prevention.

Transient receptor potential ankyrin 1 (TRPA1) channel is a calcium permeable, non-selective cation channel, expressed in the sensory neurons and non-neuronal cells of different tissues. Initially studied for its role in pain and inflammation, TRPA1 has now functionally involved in multiple other physiological functions. TRPA1 channel has been extensively studied for modulation by pungent compounds present in the spices and herbs. In the last decade, the role of TRPA1 agonism in body weight reduction, secretion of hunger and satiety hormones, insulin secretion and thermogenesis, has unveiled the potential of the TRPA1 channel to be used as a preventive target to tackle obesity and associated comorbidities including insulin resistance in type 2 diabetes. In this review, we summarized the recent findings of TRPA1 based dietary/non-dietary modulation for its role in obesity prevention and therapeutics.

Interactions between Chemesthesis and Taste: Role of TRPA1 and TRPV1.

In addition to the sense of taste and olfaction, chemesthesis, the sensation of irritation, pungency, cooling, warmth, or burning elicited by spices and herbs, plays a central role in food consumption. Many plant-derived molecules demonstrate their chemesthetic properties via the opening of transient receptor potential ankyrin 1 (TRPA1) and transient receptor potential vanilloid 1 (TRPV1) channels. TRPA1 and TRPV1 are structurally related thermosensitive cation channels and are often co-expressed in sensory nerve endings. TRPA1 and TRPV1 can also indirectly influence some, but not all, primary taste qualities via the release of substance P and calcitonin gene-related peptide (CGRP) from trigeminal neurons and their subsequent effects on CGRP receptor expressed in Type III taste receptor cells. Here, we will review the effect of some chemesthetic agonists of TRPA1 and TRPV1 and their influence on bitter, sour, and salt taste qualities.

Efficient and flexible synthesis of new photoactivatable propofol analogs.

Propofol is a widely used general anesthetic, which acts by binding to and modulating several neuronal ion channels. We describe the synthesis of photoactivatable propofol analogs functionalized with an alkyne handle for bioorthogonal chemistry. Such tools are useful for detecting and isolating photolabeled proteins. We designed expedient and flexible synthetic routes to three new diazirine-based crosslinkable propofol derivatives, two of which have alkyne handles. As a proof of principle, we show that these compounds activate heterologously expressed Transient Receptor Potential Ankyrin 1 (TRPA1), a key ion channel of the pain pathway, with a similar potency as propofol in fluorescence-based functional assays. This work demonstrates that installation of the crosslinkable and clickable group on a short nonpolar spacer at the para position of propofol does not affect TRPA1 activation, supporting the utility of these chemical tools in identifying and characterizing potentially druggable binding sites in propofol-interacting proteins.

A Non-covalent Ligand Reveals Biased Agonism of the TRPA1 Ion Channel.

The TRPA1 ion channel is activated by electrophilic compounds through the covalent modification of intracellular cysteine residues. How non-covalent agonists activate the channel and whether covalent and non-covalent agonists elicit the same physiological responses are not understood. Here, we report the discovery of a non-covalent agonist, GNE551, and determine a cryo-EM structure of the TRPA1-GNE551 complex, revealing a distinct binding pocket and ligand-interaction mechanism. Unlike the covalent agonist allyl isothiocyanate, which elicits channel desensitization, tachyphylaxis, and transient pain, GNE551 activates TRPA1 into a distinct conducting state without desensitization and induces persistent pain. Furthermore, GNE551-evoked pain is relatively insensitive to antagonist treatment. Thus, we demonstrate the biased agonism of TRPA1, a finding that has important implications for the discovery of effective drugs tailored to different disease etiologies.

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.

Gating and calcium-sensing mechanisms of TRPA1 channels revealed.

Novel structures of the human TRPA1 channel were determined in the presence of the agonist iodoacetamide and the antagonist A-967079, to reveal the open and closed states of the channel, respectively. The structures further revealed the location of Ca2+ modulatory site that is likely conserved among several TRP subgroups.

Irritant-evoked activation and calcium modulation of the TRPA1 receptor.

The transient receptor potential ion channel TRPA1 is expressed by primary afferent nerve fibres, in which it functions as a low-threshold sensor for structurally diverse electrophilic irritants, including small volatile environmental toxicants and endogenous algogenic lipids1. TRPA1 is also a 'receptor-operated' channel whose activation downstream of metabotropic receptors elicits inflammatory pain or itch, making it an attractive target for novel analgesic therapies2. However, the mechanisms by which TRPA1 recognizes and responds to electrophiles or cytoplasmic second messengers remain unknown. Here we use strutural studies and electrophysiology to show that electrophiles act through a two-step process in which modification of a highly reactive cysteine residue (C621) promotes reorientation of a cytoplasmic loop to enhance nucleophilicity and modification of a nearby cysteine (C665), thereby stabilizing the loop in an activating configuration. These actions modulate two restrictions controlling ion permeation, including widening of the selectivity filter to enhance calcium permeability and opening of a canonical gate at the cytoplasmic end of the pore. We propose a model to explain functional coupling between electrophile action and these control points. We also characterize a calcium-binding pocket that is highly conserved across TRP channel subtypes and accounts for all aspects of calcium-dependent TRPA1 regulation, including potentiation, desensitization and activation by metabotropic receptors. These findings provide a structural framework for understanding how a broad-spectrum irritant receptor is controlled by endogenous and exogenous agents that elicit or exacerbate pain and itch.

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.

Inhibition of Ligand-Gated TRPA1 by General Anesthetics.

Several general anesthetics (GAs) produce pain or irritation upon administration, and this occurs predominantly through the activation of the nociceptive ion channel, transient receptor potential ankyrin type 1 (TRPA1). However, the effects of GAs on agonist-mediated TRPA1 activity are unclear. Here we show that a diverse range of noxious and non-noxious volatile anesthetics, at clinically relevant concentrations, inhibit ligand-activated TRPA1 currents. These effects are species-specific; GAs blocks rodent TRPA1 without affecting the Drosophila ortholog. Furthermore, propofol inhibits rodent but not human TRPA1. Analysis of chimeric TRPA1 proteins and mutagenesis combined reveals two amino acid residues located in the S5 domain, Ser876 and Thr877, that are critical for the inhibitory effects of isoflurane and propofol. Introduction of these residues into Drosophila TRPA1 confers anesthetic inhibition. Furthermore, several residues lining the presumptive binding pocket for noxious GAs are not required for the inhibitory effects of GAs. We conclude that anesthetics inhibit TRPA1 by interacting at a site distinct from the activation site. The inhibitory effects of GAs at TRPA1 may contribute to the diverse pharmacological action of these drugs. SIGNIFICANCE STATEMENT: We show that both noxious and non-noxious general anesthetics inhibit agonist-evoked transient receptor potential ankyrin type 1 (TRPA1) activity and identify critical amino acid residues located in the pore domain. Inhibition of TRPA1 may affect pain and vascular signaling during anesthesia.

Calcium activates purified human TRPA1 with and without its N-terminal ankyrin repeat domain in the absence of calmodulin.

Extracellular influx of calcium or release of calcium from intracellular stores have been shown to activate mammalian TRPA1 as well as to sensitize and desensitize TRPA1 electrophilic activation. Calcium binding sites on both intracellular N- and C-termini have been proposed. Here, we demonstrate based on Förster resonance energy transfer (FRET) and bilayer patch-clamp studies, a direct calmodulin-independent action of calcium on the purified human TRPA1 (hTRPA1), causing structural changes and activation without immediate subsequent desensitization of hTRPA1 with and without its N-terminal ankyrin repeat domain (N-ARD). Thus, calcium alone activates hTRPA1 by a direct interaction with binding sites outside the N-ARD.

Semi-synthetic cinnamodial analogues: Structural insights into the insecticidal and antifeedant activities of drimane sesquiterpenes against the mosquito Aedes aegypti.

The Aedes aegypti mosquito serves as a major vector for viral diseases, such as dengue, chikungunya, and Zika, which are spreading across the globe and threatening public health. In addition to increased vector transmission, the prevalence of insecticide-resistant mosquitoes is also on the rise, thus solidifying the need for new, safe and effective insecticides to control mosquito populations. We recently discovered that cinnamodial, a unique drimane sesquiterpene dialdehyde of the Malagasy medicinal plant Cinnamosma fragrans, exhibited significant larval and adult toxicity to Ae. aegypti and was more efficacious than DEET-the gold standard for insect repellents-at repelling adult female Ae. aegypti from blood feeding. In this study several semi-synthetic analogues of cinnamodial were prepared to probe the structure-activity relationship (SAR) for larvicidal, adulticidal and antifeedant activity against Ae. aegypti. Initial efforts were focused on modification of the dialdehyde functionality to produce more stable active analogues and to understand the importance of the 1,4-dialdehyde and the α,ß-unsaturated carbonyl in the observed bioactivity of cinnamodial against mosquitoes. This study represents the first investigation into the SAR of cinnamodial as an insecticide and antifeedant against the medically important Ae. aegypti mosquito.

Identification and characterization of saikosaponins as antagonists of transient receptor potential A1 channel.

Neuropathic pain is associated with an increased sensitivity to painful stimuli or abnormal sensitivity to otherwise innocuous stimuli. However, in addition to adverse effects, currently available drugs have shown limited response in patients with neuropathic pain, which provides a rationale to explore new drug classes acting on novel targets and with better efficacy and safety profiles. Here, we found that saikosaponins potently inhibit agonist-induced activation of the transient receptor potential A1 (TRPA1) channel, which has been reported to mediate neuropathic pain by sensing a variety of chemical irritants. Molecular docking and site-directed mutagenesis analyses suggested that saikosaponins bind to the hydrophobic pocket in TRPA1 near the Asn855 residue, which, when mutated to Ser, was previously associated with enhanced pain perception in humans. In support of these findings, saikosaponin D significantly attenuated agonist-induced nociceptive responses and vincristine-induced mechanical hypersensitivity in mice. These results indicate that saikosaponins are TRPA1 antagonists and provide a basis for further elaboration of saikosaponin derivatives for the development of new therapeutics for neuropathic pain.

TRPA1 and issues relating to animal model selection for extrapolating toxicity data to humans.

The transient receptor potential ankyrin 1 (TRPA1) ion channel is a sensor for irritant chemicals, has ancient lineage, and is distributed across animal species including humans, where it features in many organs. Its activation by a diverse panel of electrophilic molecules (TRPA1 agonists) through electrostatic binding and/or covalent attachment to the protein causes the sensation of pain. This article reviews the species differences between TRPA1 channels and their responses, to assess the suitability of different animals to model the effects of TRPA1-activating electrophiles in humans, referring to common TRPA1 activators (exogenous and endogenous) and possible mechanisms of action relating to their toxicology. It concludes that close matching of in vitro and in vivo models will help optimise the identification of relevant biochemical and physiological responses to benchmark the efficacy of potential therapeutic drugs, including TRPA1 antagonists, to counter the toxic effects of those electrophiles capable of harming humans. The analysis of the species issue provided should aid the development of medical treatments to counter poisoning by such chemicals.

Diverse sensitivities of TRPA1 from different mosquito species to thermal and chemical stimuli.

Temperature and odors profoundly affect the behavior of animals. Transient receptor potential channel, subfamily A, member 1 (TRPA1) functions as a polymodal nociceptor for sensing both vital environmental cues in insects. Mosquitoes are recognized as disease vectors, and many efforts have been devoted to investigations of their host-seeking behaviors and repellents. However, the physiological characteristics of mosquito TRPA1 have not been systematically studied. We identified multiple alternative splice variants of the TrpA1 gene from Anopheles gambiae, Anopheles stephensi, Aedes aegypti and Culex pipiens pallens mosquitoes. And we performed comparative analyses of the responses of mosquito TRPA1s to heat or chemical stimuli with calcium-imaging and whole-cell patch-clamp methods. Comparison of TRPA1 among four mosquito species from different thermal niches revealed that TRPA1 of Culex pipiens pallens inhabiting the temperate zone had a lower temperature threshold for heat-evoked activation, which was supported by the in vivo heat-avoidance test. Notably, the chemosensitivity of mosquito TRPA1 channels revealed differences not only between variants but also among species. Moreover, we discovered three novel mosquito TRPA1 agonists. Thermal niches selection and evolutionary trajectories significantly affect the functional properties of mosquito TRPA1, which represents a hallmark of the behaviors that may permit the design of improved mosquito control methods.

TRPA1 modulation by piperidine carboxamides suggests an evolutionarily conserved binding site and gating mechanism.

The transient receptor potential ankyrin 1 (TRPA1) channel functions as an irritant sensor and is a therapeutic target for treating pain, itch, and respiratory diseases. As a ligand-gated channel, TRPA1 can be activated by electrophilic compounds such as allyl isothiocyanate (AITC) through covalent modification or activated by noncovalent agonists through ligand binding. However, how covalent modification leads to channel opening and, importantly, how noncovalent binding activates TRPA1 are not well-understood. Here we report a class of piperidine carboxamides (PIPCs) as potent, noncovalent agonists of human TRPA1. Based on their species-specific effects on human and rat channels, we identified residues critical for channel activation; we then generated binding modes for TRPA1-PIPC interactions using structural modeling, molecular docking, and mutational analysis. We show that PIPCs bind to a hydrophobic site located at the interface of the pore helix 1 (PH1) and S5 and S6 transmembrane segments. Interestingly, this binding site overlaps with that of known allosteric modulators, such as A-967079 and propofol. Similar binding sites, involving π-helix rearrangements on S6, have been recently reported for other TRP channels, suggesting an evolutionarily conserved mechanism. Finally, we show that for PIPC analogs, predictions from computational modeling are consistent with experimental structure-activity studies, thereby suggesting strategies for rational drug design.