TRPA1 Channels Research Articles

TRPA1-dependent and -independent activation by commonly used preservatives.

Background and purpose: Addition of preservatives ensures microbial stability, especially in multidose containers of parenterally administered pharmaceuticals. These compounds can cause side effects, and particularly at the site of application, might elicit or facilitate pain. TRPA1 is a cation channel expressed in peripheral neurons which contributes to pain and inflammation and is sensitive to many irritants. The most commonly used preservatives, in particular with a focus on parenteral formulations, were investigated for their potential to activate TRPA1. Experimental approach: Sixteen preservatives were screened for mediating calcium influx in human TRPA1-transfected HEK293t cells. Untransfected cells served as control, results were further validated in mouse sensory neurons. In addition, proinflammatory mediators serotonin, histamine and prostaglandin E2 were co-administered to probe a potential sensitisation of preservative-induced TRPA1 activation. Key results: Butylparaben, propylparaben, ethylparaben, bronopol, methylparaben, phenylethyl alcohol and phenol induced a TRPA1-dependent calcium influx in transfected HEK293t cells at concentrations used for preservation. Other preservatives increased calcium within the used concentration ranges, but to a similar degree in untransfected controls. Serotonin, histamine, and prostaglandin enhanced TRPA1 activation of phenylethyl alcohol, bronopol, ethylparaben, propylparaben and butylparaben. Conclusion and implications: Systematic screening of common preservatives applied for parenterally administered drugs resulted in identifying several preservatives with substantial TRPA1 channel activation. This activation was enhanced by the addition of proinflammatory meditators. This allows selecting a preservative without TRPA1 activation, particularly in case of pharmaceuticals that could act proinflammatory.

Zinc pyrithione induces endothelium-dependent hyperpolarization-mediated mesenteric vasorelaxation in healthy and colitic mice

BackgroundAlthough Zinc pyrithione (ZPT) could lower blood pressure by inducing vasorelaxation, it is unclear if it is able to induce vasorelaxation of mesenteric arterioles in health and ulcerative colitis (UC) to exert anti-colitic action. MethodsThe vasorelaxation of the second-order branch of the mesenteric artery from wide type (WT) mice, TRPV1−/−(KO) mice, and TRPV4−/−(KO) mice was determined using a Mulvany-style wire myograph. Calcium imaging and patch clamp were applied to analyze the actions of ZPT in human vascular endothelial cells. Mouse model of UC was used to evaluate the anti-colitic action of ZPT. ResultsZPT dose-dependently induced mesenteric vasorelaxation predominately through endothelium-dependent hyperpolarization (EDH), which could be attenuated by intracellular Zn2+ and Ca2+ chelators TPEN and BAPTA-AM. The ZPT/EDH-mediated vasorelaxation via TRPV1, TRPV4 and TRPA1 channels was verified by a combination of selective pharmacological inhibitors and TRPV1-KO and TRPV4-KO mice. Moreover. ZPT induced Ca2+ entry via vascular endothelial TRPV1/4 and TRPA1 channels and enhanced membrane non-selective currents through these channels. Notably, ZPT exerted anti-colitic effects by rescuing the impaired acetylcholine (ACh)-induced mesenteric vasorelaxation in colitic mice. ConclusionsZPT/Zn2+ induces EDH-mediated mesenteric vasorelaxation through activating endothelial multiple TRPV1/4 and TPPA1 channels in health, and rescues the impaired ACh-induced vasorelaxation to exert anti-colitic action. Our study may open a new avenue of potential vessel-specific targeted therapy for UC.

Functional dynamics and allosteric modulation of TRPA1

The cation channel TRPA1 is a potentially important drug target, and characterization of TRPA1 functional dynamics might help guide structure-based drug design. Here, we present results from long-timescale molecular dynamics simulations of TRPA1 with an allosteric activator, allyl isothiocyanate (AITC), in which we observed spontaneous transitions from a closed, non-conducting channel conformation into an open, conducting conformation. Based on these transitions, we propose a gating mechanism in which movement of a regulatory TRP-like domain allosterically translates into pore opening in a manner reminiscent of pore opening in voltage-gated ion channels. In subsequent experiments, we found that mutations that disrupt packing of the S4-S5 linker-TRP-like domain and the S5 and S6 helices also affected channel activity. In simulations, we also observed A-967079, a known allosteric inhibitor, binding between helices S5 and S6, suggesting that A-967079 may suppress activity by stabilizing a non-conducting pore conformation-a finding consistent with our proposed gating mechanism.

The conducting state of TRPA1 modulates channel lateral mobility

We have studied Danio rerio (Zebrafish) TRPA1 channel using a method that combines single channel electrophysiological and optical recordings to evaluate lateral mobility and channel gating simultaneously in single channels. TRPA1 channel activation by two distinct chemical ligands: allyl isothiocyanate (AITC) and TRPswitch B, results in substantial reduction of channel lateral mobility at the plasma membrane. Incubation with the cholesterol sequestering agent methyl-β-cyclodextrin (MβCD), prevents the reduction on lateral mobility induced by the two chemical agonists. This results strongly suggest that the open conformation of TRPA1 modulates channel lateral mobility probably by facilitating the insertion of the channel into cholesterol-enriched domains at the plasma membrane.

EGb 761 reduces Ca2+ influx and apoptosis after pentylenetetrazole treatment in a neuroblastoma cell line.

Transient receptor potential (TRP) channels have been found to have significant implications in neuronal outgrowth, survival, inflammatory neurogenic pain, and various epileptogenic processes. Moreover, there is a growing body of evidence indicating that transient receptor potential (TRP) channels have a significant impact on epilepsy and its drug-resistant subtypes. We postulated that EGb 761 would modulate TRPA1 channels, thereby exhibiting anti-inflammatory and neuroprotective effects in a neuroblastoma cell line. Our rationale was to investigate the impact of EGb 761 in a controlled model of pentylenetetrazole-induced generalized epilepsy. We evaluated the neuroprotective, antioxidant and anti-apoptotic effects of EGb 761 both before and after the pentylenetetrazole application in a neuroblastoma cell line. Specifically, we focused on the effects of EGB 761 on the activity of Transient receptor potential (TRP) channels. EGb 761 applications both before and after the pentylenetetrazole incubation period reduced Ca release and restored apoptosis, ROS changes, mitochondrial depolarization and caspase levels, suggesting a prominent prophylactic and therapeutic effect of EGb 761 in the pentylenetetrazole-induced epileptogenesis process. Our basic mechanistic framework for elucidating the pathophysiological significance of fundamental ion mechanisms in a pentylenetetrazole treated neuroblastoma cell line provided compelling evidence for the favorable efficacy and safety profile of Egb 761 in human-relevant in vitro model of epilepsy. To the best of our knowledge, this is the first study to investigate the combined effects of EGb 761 and pentylenetetrazole on TRP channels and measure their activation level in a relevant model of human epileptic diseases.

Allyl isothiocyanate mitigates airway inflammation and constriction in a house dust mite-induced allergic asthma model via upregulation of tight junction proteins and the TRPA1 modulation

Asthma is a chronic inflammatory disease that has been associated with insufficient vegetable intake. Allyl Isothiocyanate (AITC) is a natural isothiocyanate found in cruciferous plants with anti-inflammatory and antioxidant abilities. Our study aimed to investigate the potential effect of AITC on tracheal constriction in a house dust mite (HDM)-induced asthma animal model, and explore the underlying mechanisms. To investigate the effects of AITC on HDM-induced allergic asthma model, established by intranasally administering extracts of HDM and AITC or DEX was given orally for four weeks. Flexivent SCIREQ, H&E staining, ELISA were employed to evaluate the lung function and the cytokine secretion. Possible mechanisms were determined by Western blot. Rat tracheae contraction was measured by Labscribe. We utilized lung epithelial cells (BEAS-2B) to assess the adhesion response to the combination of inflammatory factors TNF-α and IL-4. The results of the study showed that AITC significantly reduced tracheal constriction in ex vivo experiments and improved lung function in in vivo experiments compared to HDM-induced mice. Additionally, AITC decreased cytokine secretion, inflammatory cell infiltration in the lung, and constriction-related proteins expression in both lung and tracheae. Moreover, AITC increased tight junction-related protein expression in lung tissues. In vitro experiments showed that AITC had a protective effect through TRPA1 channel without affecting cell viability. Our results demonstrate that AITC has potential anti-asthma effects in HDM-induced asthma models by alleviating airway inflammation and airway constriction through increasing tight junction-related protein expression and suppressing Ca2+ signaling. These findings suggest that AITC may be a beneficial adjuvant therapy in asthma treatment.

TRPA1 and TPRV1 Ion Channels Are Required for Contact Lens-Induced Corneal Parainflammation and Can Modulate Levels of Resident Corneal Immune Cells.

Contact lens wear can induce corneal parainflammation involving CD11c+ cell responses (24 hours), γδ T cell responses (24 hours and 6 days), and IL-17-dependent Ly6G+ cell responses (6 days). Topical antibiotics blocked these CD11c+ responses. Because corneal CD11c+ responses to bacteria require transient receptor potential (TRP) ion-channels (TRPA1/TRPV1), we determined if these channels mediate lens-induced corneal parainflammation. Wild-type mice were fitted with contact lenses for 24 hours or 6 days and compared to lens wearing TRPA1 (-/-) or TRPV1 (-/-) mice or resiniferatoxin (RTX)-treated mice. Contralateral eyes were not fitted with lenses. Corneas were examined for major histocompatibility complex (MHC) class II+, CD45+, γδ T, or TNF-α+ cell responses (24 hours) or Ly6G+ responses (6 days) by quantitative imaging. The quantitative PCR (qPCR) determined cytokine gene expression. Lens-induced increases in MHC class II+ cells after 24 hours were abrogated in TRPV1 (-/-) but not TRPA1 (-/-) mice. Increases in CD45+ cells were unaffected. Increases in γδ T cells after 24 hours of wear were abrogated in TRPA1 (-/-) and TRPV1 (-/-) mice, as were 6 day Ly6G+ cell responses. Contralateral corneas of TRPA1 (-/-) and TRPV1 (-/-) mice showed reduced MHC class II+ and γδ T cells at 24 hours. RTX inhibited lens-induced parainflammatory phenotypes (24 hours and 6 days), blocked lens-induced TNF-α and IL-18 gene expression, TNF-α+ cell infiltration (24 hours), and reduced baseline MHC class II+ cells. TRPA1 and TRPV1 mediate contact lens-induced corneal parainflammation after 24 hours and 6 days of wear and can modulate baseline levels of resident corneal immune cells.

TRPA1 activation in non-sensory supporting cells contributes to regulation of cochlear sensitivity after acoustic trauma

TRPA1 channels are expressed in nociceptive neurons, where they detect noxious stimuli, and in the mammalian cochlea, where their function is unknown. Here we show that TRPA1 activation in the supporting non-sensory Hensen’s cells of the mouse cochlea causes prolonged Ca2+ responses, which propagate across the organ of Corti and cause long-lasting contractions of pillar and Deiters’ cells. Caged Ca2+ experiments demonstrated that, similar to Deiters’ cells, pillar cells also possess Ca2+-dependent contractile machinery. TRPA1 channels are activated by endogenous products of oxidative stress and extracellular ATP. Since both these stimuli are present in vivo after acoustic trauma, TRPA1 activation after noise may affect cochlear sensitivity through supporting cell contractions. Consistently, TRPA1 deficiency results in larger but less prolonged noise-induced temporary shift of hearing thresholds, accompanied by permanent changes of latency of the auditory brainstem responses. We conclude that TRPA1 contributes to the regulation of cochlear sensitivity after acoustic trauma.

Thermal hyperalgesia and mechanical allodynia in itch TRPA1 and TRPV1 channels

Thermal hyperalgesia and mechanical allodynia in itch TRPA1 and TRPV1 channels

Functional expression of the thermally activated transient receptor potential channels TRPA1 and TRPM8 in human myotubes

Transient potential (TRP) ion channels expressed in primary sensory neurons act as the initial detectors of environmental cold and heat, information which controls muscle energy expenditure. We hypothesize that non-neuronal TRPs have direct cellular responses to thermal exposure, also affecting cellular metabolism. In the present study we show expression of TRPA1, TRPM8 and TRPV1 in rat skeletal muscle and human primary myotubes by qPCR. Effects of TRP activity on metabolism in human myotubes were studied using radiolabeled glucose. FURA-2 was used for Ca2+ imaging.TRPA1, TRPM8 and TRPV1 were expressed at low levels in primary human myotubes and in m. gastrocnemius, m. soleus, and m. trapezius from rat. Activation of TRPA1 by ligustilide resulted in an increased glucose uptake and oxidation in human myotubes, whereas activation of TRPM8 by menthol and icilin significantly decreased glucose uptake and oxidation. Activation of heat sensing TRPV1 by capsaicin had no effect on glucose metabolism. Agonist-induced increases in intracellular Ca2+ levels by ligustilide and icilin in human myotubes confirmed a direct activation of TRPA1 and TRPM8, respectively. The mRNA expression of some genes involved in thermogenesis, i.e. peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), uncoupling protein (UCP) 1 and UCP3, were downregulated in human myotubes following TRPA1 activation, while the mRNA expression of TRPM8 and TRPA1 were downregulated following TRPM8 activation by menthol and icilin, respectively. Cold exposure (18 °C) of cultured myotubes followed by a short recovery period had no effect on glucose uptake and oxidation in the basal situation, however when TRPA1 and TRPM8 channels were chemically inhibited a temperature-induced difference in glucose metabolism was found.In conclusion, mRNA of TRPA1, TRPM8 and TRPV1 are expressed in rat skeletal muscle and human skeletal muscle cells. Modulation of TRPA1 and TRPM8 by chemical agents induced changes in Ca2+ levels and glucose metabolism in human skeletal muscle cells, indicating functional receptors.

Topical borneol relieves nonhistaminergic pruritus via targeting TRPA1 and TRPM8 channels in peripheral nerve terminals of mice

Borneol has been used successfully for the treatment of itchy skin in traditional Chinese medicine. However, the antipruritic effect of borneol has rarely been studied, and the mechanism is unclear. Here, we showed that topical application of borneol on skin substantially suppressed pruritogen chloroquine- and compound 48/80-induced itching in mice. The potential targets of borneol, including transient receptor potential cation channel subfamily V member 3 (TRPV3), transient receptor potential cation channel subfamily A member 1 (TRPA1), transient receptor potential cation channel subfamily M member 8 (TRPM8), and gamma-aminobutyric acid type A (GABAA) receptor were pharmacologically inhibited or genetically knocked out one by one in mouse. Itching behavior studies demonstrated that the antipruritic effect of borneol is largely independent of TRPV3 and GABAA receptor, and TRPA1 and TRPM8 channels are responsible for a major portion of the effect of borneol on chloroquine-induced nonhistaminergic itching. Borneol activates TRPM8 and inhibits TRPA1 in sensory neurons of mice. Topical co-application of TRPA1 antagonist and TRPM8 agonist mimicked the effect of borneol on chloroquine-induced itching. Intrathecal injection of a group II metabotropic glutamate receptor antagonist partially attenuated the effect of borneol and completely abolished the effect of TRPM8 agonist on chloroquine-induced itching, suggesting that a spinal glutamatergic mechanism is involved. In contrast, the effect of borneol on compound 48/80-induced histaminergic itching occurs through TRPA1-and TRPM8-independent mechanisms. Our work demonstrates that borneol is an effective topical itch reliever, and TRPA1 inhibition and TRPM8 activation in peripheral nerve terminals account for its antipruritic effect.

Recombinant Production, NMR Solution Structure, and Membrane Interaction of the Phα1β Toxin, a TRPA1 Modulator from the Brazilian Armed Spider Phoneutria nigriventer.

Phα1β (PnTx3-6) is a neurotoxin from the spider Phoneutria nigriventer venom, originally identified as an antagonist of two ion channels involved in nociception: N-type voltage-gated calcium channel (CaV2.2) and TRPA1. In animal models, Phα1β administration reduces both acute and chronic pain. Here, we report the efficient bacterial expression system for the recombinant production of Phα1β and its 15N-labeled analogue. Spatial structure and dynamics of Phα1β were determined via NMR spectroscopy. The N-terminal domain (Ala1-Ala40) contains the inhibitor cystine knot (ICK or knottin) motif, which is common to spider neurotoxins. The C-terminal α-helix (Asn41-Cys52) stapled to ICK by two disulfides exhibits the µs-ms time-scale fluctuations. The Phα1β structure with the disulfide bond patterns Cys1-5, Cys2-7, Cys3-12, Cys4-10, Cys6-11, Cys8-9 is the first spider knottin with six disulfide bridges in one ICK domain, and is a good reference to other toxins from the ctenitoxin family. Phα1β has a large hydrophobic region on its surface and demonstrates a moderate affinity for partially anionic lipid vesicles at low salt conditions. Surprisingly, 10 µM Phα1β significantly increases the amplitude of diclofenac-evoked currents and does not affect the allyl isothiocyanate (AITC)-evoked currents through the rat TRPA1 channel expressed in Xenopus oocytes. Targeting several unrelated ion channels, membrane binding, and the modulation of TRPA1 channel activity allow for considering Phα1β as a gating modifier toxin, probably interacting with S1-S4 gating domains from a membrane-bound state.

Melatonin promotes orofacial antinociception in adult zebrafish by modulating TRP channels

Melatonin is an endogenous hormone, known as the sleep hormone, which has already demonstrated its antinociceptive effect. This study aimed to evaluate the participation of TRP's channels in the orofacial antinociceptive effect of melatonin (MT) in adult zebrafish. Initially, the open field test was performed to evaluate the effect of MT on the locomotor activity of adult zebrafish. Then, the animals were pre-treated with MT (0.1, 0.3 or 1 mg/mL; gavage) and acute orofacial nociception was induced by the application of capsaicin (TRPV1 agonist), cinnamaldehyde (TRPA1 agonist) or menthol (TRPM8 agonist) applied into the animal's lip. Naive groups were included. MT, per se, did not alter the locomotor activity of the animals. MT reduced the nociceptive behavior induced by the three agonists; however, the most significant effect was obtained with the lowest concentration tested (0.1 mg/mL) in the capsaicin test. The orofacial antinociceptive effect of melatonin was prevented by capsazepine (TRPV1 antagonist), but not by HC-030031 (TRPA1 antagonist). The molecular docking study indicated interaction between MT and the TRPV1, TRPA1 and TRPM8 channels and, in line with the in vivo results, there was greater affinity between MT and the TRPV1 channel. The results confirm the pharmacological relevance of melatonin as an inhibitor of orofacial nociception and this effect seems to be related to the modulation of TRP's channels.

TRPA1 is involved in the inhibitory effect of Ke-teng-zi on allergic contact dermatitis via MAPK and JAK/STAT3 signaling pathways.

The seeds of Entada phaseoloides (Linn.) Merr. commonly named "Ke-teng-zi" is a traditional Chinese folk medicine and reported to treat dermatitis, spasm, and headache. However, the exact effect and the mechanism of Ke-teng-zi on the treatment of dermatitis is unclear. To elucidate the antipruritic effect and molecular mechanisms of Ke-teng-zi on the treatment of allergic contact dermatitis (ACD). The main components of the n-butanol fraction of 70% ethanol extract from Ke-teng-zi (abbreviated as KB) were analyzed by HPLC. The chloroquine (CQ)-induced acute itch and squaraine dibutyl ester (SADBE)-induced ACD chronic itch in mice was established, and the TNF-α/IFN-γ stimulated Human keratinocytes (HaCaT) were used to evaluate the antipruritic and anti-inflammatory effects of KB. Behavioral tests, lesion scoring, and histology were also examined. The expression levels of molecules in MAPK and JAK/STAT3 pathways, the mRNA levels of chemokines and cytokines in both the skin of ACD mice and the HaCaT cells were detected by western blot and qPCR. Furthermore, whole-cell patch-clamp recordings in TRPA1-tranfected HEK293T cells were used to elucidate the effect of KB on TRPA1 channels. TRPA1 siRNA was used to evaluate the role of TRPA1 in the anti-inflammatory effect of KB in keratinocytes. The main compounds in KB could bind to the active sites of TRPA1 mainly through hydrogen bond and hydrophobic bond interactions. KB could inhibit the scratching behavior in CQ-induced acute itch, and the inhibitory effect of KB was blocked by TRPA1 inhibitor HC-030031. In addition, KB significantly decreased the scratching bouts of ACD mice, reduced the skin lesion scores, mast cells degranulation, and epidermal thickening, inhibited the production of inflammatory chemokines/cytokines and CGRP, and down-regulated the levels of p-ERK1/2, p-p38, and p-STAT3, compared to the ACD mice. Moreover, continuous application of KB induced the desensitization of TRPA1 channels. Also, KB inhibited the expression of p-ERK1/2, p-p38, and p-STAT3, and down-regulated the expression of inflammatory chemokines and cytokines in vitro, which were reversed by the TRPA1 siRNA. KB alleviated the pruritus and skin inflammation in ACD mice through TRPA1 channels desensitization and down-regulation of intracellular MAPK and JAK/STAT3 signaling pathways. Our results suggested that Ke-teng-zi is a potential drug for the treatment of inflammatory skin diseases such as ACD.

Molecular mechanisms of MrgprA3-independent activation of the transient receptor potential ion channels TRPA1 and TRPV1 by chloroquine.

Itch associates several pathologies and is a common drug-induced side effect. Chloroquine (CQ) was reported to induce itch by activating the Mas-related G protein-coupled receptor MrgprA3 and subsequently TRPA1. In this study we demonstrate that CQ employs at least two MrgprA3-independent mechanisms to activate or sensitize TRPA1 and TRPV1. Patch Clamp and calcium-imaging were utilized to examine effects of CQ on TRPA1 and TRPV1 expressed in HEK-293T cells. In calcium-imaging, CQ induces a concentration-dependent but MrgprA3-independent activation of TRPA1 and TRPV1. While CQ itself inhibits TRPA1 and TRPV1 in patch clamp recordings, co-application of CQ and UVA-light evokes membrane currents through both channels. This effect is inhibited by the reducing agent dithiothreitol (DTT) and reduced on mutants lacking cysteine residues accounting for reactive oxygen species (ROS)-sensitivity. The combination of CQ and UVA-light triggers an accumulation of intracellular ROS, removes fast inactivation of voltage-gated sodium currents and activates TRPV2. On the other hand, CQ is a weak base and induces intracellular alkalosis. Intracellular alkalosis can activate TRPA1 and TRPV1, and CQ applied at alkaline pH-values indeed activates both channels. Our data reveal novel pharmacological properties of CQ allowing activation of TRPA1 and TRPV1 via photosensitization as well as intracellular alkalosis. These findings add complexity to the commonly accepted dogma that CQ-induced itch is specifically mediated by MrgprA3 coupling to TRPA1.

An Optochemical Oxygen Scavenger Enabling Spatiotemporal Control of Hypoxia.

We present an optochemical O2 scavenging system that enables precise spatiotemporal control of the level of hypoxia in living cells simply by adjusting the light intensity in the illuminated region. The system employs rhodamine containing a selenium or tellurium atom as an optochemical oxygen scavenger that rapidly consumes O2 by photochemical reaction with glutathione as a coreductant upon visible light irradiation (560-590 nm) and has a rapid response time, within a few minutes. The glutathione-consuming quantum yields of the system were calculated as about 5 %. The spatiotemporal O2 consuming in cultured cells was visualized with a hypoxia-responsive fluorescence probe, MAR. Phosphorescence lifetime imaging was applied to confirmed that different light intensities could generate different levels of hypoxia. To illustrate the potential utility of this system for hypoxia research, we show that it can spatiotemporally control calcium ion (Ca2+ ) influx into HEK293T cells expressing the hypoxia-responsive Ca2+ channel TRPA1.

An Optochemical Oxygen Scavenger Enabling Spatiotemporal Control of Hypoxia

AbstractWe present an optochemical O2 scavenging system that enables precise spatiotemporal control of the level of hypoxia in living cells simply by adjusting the light intensity in the illuminated region. The system employs rhodamine containing a selenium or tellurium atom as an optochemical oxygen scavenger that rapidly consumes O2 by photochemical reaction with glutathione as a coreductant upon visible light irradiation (560–590 nm) and has a rapid response time, within a few minutes. The glutathione‐consuming quantum yields of the system were calculated as about 5 %. The spatiotemporal O2 consuming in cultured cells was visualized with a hypoxia‐responsive fluorescence probe, MAR. Phosphorescence lifetime imaging was applied to confirmed that different light intensities could generate different levels of hypoxia. To illustrate the potential utility of this system for hypoxia research, we show that it can spatiotemporally control calcium ion (Ca2+) influx into HEK293T cells expressing the hypoxia‐responsive Ca2+ channel TRPA1.

Molecular architecture and gating mechanisms of the Drosophila TRPA1 channel

The transient receptor potential channel subfamily A member 1 (TRPA1) ion channel is an evolutionary conserved polymodal sensor responding to noxious temperature or chemical stimuli. Notably, the thermosensitivity of TRPA1 varies among different species or even different isoforms in the same species. However, the underlying molecular basis of its thermo-gating remains largely unknown. Here, we determine the structures of a heat-sensitive isoform of TRPA1 in Drosophila melanogaster in two distinct conformations with cryo-samples prepared at 8 °C. Large conformational changes are observed in the ankyrin repeat domain (ARD) and the coiled-coil domain between the two states. Remarkably, all 17 ankyrin repeats are mapped in the newly resolved conformation, forming a propeller-like architecture. Two intersubunit interfaces are identified in the amino (N)-terminal domain, and play vital roles during both heat and chemical activation as shown by electrophysiological analysis. With cryo-samples prepared at 35 °C, only one conformation is resolved, suggesting possible state transitions during heat responses. These findings provide a basis for further understanding how the ARD regulates channel functions, and insights into the gating mechanism of TRPA1.

Phytol from Faeces Bombycis alleviated migraine pain by inhibiting Nav1.7 sodium channels.

Faeces Bombycis (silkworm excrement, called Cansha in Chinese), is the dried faeces of the larvae of silkworm. According to the theories of traditional Chinese medicine recorded in "Compendium of Materia Medica", Faeces Bombycis has often been prescribed in traditional Chinese medicine for the treatment of recurrent headache, rheumatalgia, rubella and itching et al. However, the bioactive components and their exact mechanisms underlying the pain-relieving effects remain to be revealed. The present study aimed to evaluate the analgesic effect of Faeces Bombycis extract (FBE) on migraine, explore the main active constituents and investigate the pharmacological mechanisms for its pain relief. The bioactivity of different extracts from Faeces Bombycis was tracked by the nitroglycerin (NTG)-induced migraine model on rats and identified by NMR spectroscopic data. Whole-cell patch clamp technique, an electrophysiological method, was used to screen the potential targets and study the mechanism of action for the bioactive compound. The following targets have been screened and studied, including Nav1.7 sodium channels, Nav1.8 sodium channels, TRPV1 channels and TRPA1 channels. The trigeminal ganglion neurons were further used to study the effects of the identified compound on neuronal excitability. By testing the bioactivity of the different extracts proceedingly, fraction petroleum ether showed higher anti-migraine activity. Through further step-by-step isolations, 7 compounds were isolated. Among them, phytol was identified with the highest yield and displayed a potent anti-migraine effect. By screening the potential ion channel targets for migraine, phytol was found to preferentially block the inactivated state of Nav1.7 sodium channels with half-inhibition concentration 0.32±0.05μM. Thus, the effects of phytol on the biophysical properties of Nav1.7 sodium channels were further characterized. Phytol induced a hyperpolarizing shift of voltage-dependent inactivation and slowed the recovery from inactivation. The affinity of phytol became weaker in the inactivation-deficient Nav1.7 channels (Nav1.7-WCW). And such an effect was independent on the local anesthetic site (Nav1.7 F1737A). Consistent with the data from recombinant channels, the compound also displayed state-dependent inhibition on neuronal sodium channels and further decreased the neuronal excitability in trigeminal ganglion neurons. Moreover, besides Nav1.7 channel, phytol also antagonized the activation of TRPV1 and TRPA1 channels at micromolar concentrations with a weaker affinity. Our results demonstrated that phytol is the major anti-migraine ingredient of Faeces Bombycis and alleviates migraine behaviors by acting on Nav1.7 sodium channels in the trigeminal ganglion neurons. This study provided evidences for the therapeutic application of Faeces Bombycis and phytol on migraine disease.

Multiplex epigenome editing of ion channel expression in nociceptive neurons abolished degenerative IVD-conditioned media-induced mechanical sensitivity.

Low back pain is a major contributor to disability worldwide and generates a tremendous socioeconomic impact. The degenerative intervertebral disc (IVD) has been hypothesized to contribute to discogenic pain by sensitizing nociceptive neurons innervating the disc to stimuli that is nonpainful in healthy patients. Previously, we demonstrated the ability of degenerative IVDs to sensitize neurons to mechanical stimuli; however, elucidation of degenerative IVDs discogenic pain mechanisms is required to develop therapeutic strategies that directly target these mechanisms. In this study, we utilized CRISPR epigenome editing of nociceptive neurons to identify mechanisms of degenerative IVD-induced changes to mechanical nociception and demonstrated the ability of multiplex CRISPR epigenome editing of nociceptive neurons to modulate inflammation-induced mechanical nociception. Utilizing an in vitro model, we demonstrated degenerative IVD-produced IL-6-induced increases in nociceptive neuron activity in response to mechanical stimuli, mediated by TRPA1, ASIC3, and Piezo2 ion channel activity. Once these ion channels were identified as mediators of degenerative IVD-induced mechanical nociception, we developed singleplex and multiplex CRISPR epigenome editing vectors that modulate endogenous expression of TRPA1, ASIC3, and Piezo2 via targeted gene promoter histone methylation. When delivered to nociceptive neurons, the multiplex CRISPR epigenome editing vectors abolished degenerative IVD-induced mechanical nociception while preserving nonpathologic neuron activity. This work demonstrates the potential of multiplex CRISPR epigenome editing as a highly targeted gene-based neuromodulation strategy for the treatment of discogenic pain, specifically; and, for the treatment of inflammatory chronic pain conditions, more broadly.