Cation Channel TRPA1 Research Articles

Photochemical activation of TRPA1 channels in neurons and animals

Optogenetics is a powerful research tool because it enables high-resolution optical control of neuronal activity. However, current optogenetic approaches are limited to transgenic systems expressing microbial opsins and other exogenous photoreceptors. Here, we identify optovin, a small molecule that enables repeated photoactivation of motor behaviors in wild type animals. Surprisingly, optovin's behavioral effects are not visually mediated. Rather, photodetection is performed by sensory neurons expressing the cation channel TRPA1. TRPA1 is both necessary and sufficient for the optovin response. Optovin activates human TRPA1 via structure-dependent photochemical reactions with redox-sensitive cysteine residues. In animals with severed spinal cords, optovin treatment enables control of motor activity in the paralyzed extremities by localized illumination. These studies identify a light-based strategy for controlling endogenous TRPA1 receptors in vivo, with potential clinical and research applications in non-transgenic animals, including humans.

Umbellulone modulates TRP channels

Inhalation of umbellulone (UMB), the offensive principle of the so-called "headache tree" (California bay laurel, Umbellularia californica Nutt.), causes a painful cold sensation. We therefore studied the action of UMB and some derivatives devoid of thiol-trapping properties on the "cold" transient receptor potential cation channels TRPA1 and TRPM8. UMB activated TRPA1 in a dose-dependent manner that was attenuated by cysteine-to-serine isosteric mutation in TRPA1 (C622S), while channel block was observed at higher concentration. However, although activation by mustard oil was completely prevented in these mutants, UMB still retained activating properties, indicating that it acts on TRPA1 only as a partial electrophilic agonist. UMB also activated TRPM8, but to a lower extent than TRPA1. Removing Michael acceptor properties of UMB (reduction or nucleophilic trapping) was detrimental for the activation of TRPA1, but increased the blocking potency. This was, however, attenuated by acetylation of the hydroxylated analogs. All UMB derivatives, except the acetylated derivatives, were also TRPM8 activators. They acted, however, in a bimodal manner, inhibiting the channel more potently than UMB, and with tetrahydro-UMB being the most potent TRPM8 activator. In conclusion, UMB is a bimodal activator of TRPA1 and a weak activator of TRPM8. Non-electrophilic derivatives of UMB are better TRPM8 activators than the natural product and also potent blockers of this channel as well as of TRPA1. The lack of effects of the acetylated UMB derivatives suggests that steric hindrance may prevent access to the recognition site for the bicyclic monoterpene pharmacophore on TRPA1 and TRPM8.

Assessment of Mucosal Movement in the Gastroesophageal Junction High-Pressure Zone During Swallowing Using Simultaneous Endoluminal Ultrasound and Manometry

BACKGROUND & AIM: Allyl isothiocyanate, the pungent ingredient of wasabi and mustard oil, is known to activate transient receptor potential (TRP) cation channels TRPA1. TRPA1 was recently shown to be functionally expressed in the gastrointestinal tract, whose ion channel presents in a subpopulation of primary sensory neurons that coexpressed with TRPV1. Previously, we also reported (1) that TRPV1-expressing sensory nerve fivers abundantly exist around blood vessels in the submucosa in the rat stomach, suggesting that TRPV1 plays an important role in control of local blood flow (Scand J. Gastroenterol. 39, 303-312, 2004), and (2) that allyl isothiocyanate facilitates to increase gastric mucosal blood flow (GMBF) in the rat stomach (Gastroenterology 138 (Suppl.1) S-721, 2010). However, the mechanism for the increased GMBF in response to allyl isothiocyanate remains unknown. In the present studies, we examined the effect of a TRPA1 activator allyl isothiocyanate on GMBF in the ex-vivo stomach of normal and sensory deafferented rats. METHODS: Male SD rats (160-200 g) were used after 18 hr-fasting. GMBFwasmeasured in the ex-vivo stomach of urethane-anesthetized rats by using the laser Doppler flowmeter. Allyl isothiocyanate (0.3310 mM) or a TRPV1 channel activator capsaicin (3.3 mM) was topically applied for 10 min to the rat stomach, and changes in GMBF were monitored. Either a TRPA1 channel blocker HC-030031 (14.1 mM) or a TRPV1 channel blocker BCTC (0.8 mM) was co-applied with allyl isothiocyanate (10 mM) for 10 min to the stomach. RESULTS: Mucosal application of allyl isothiocyanate increased GMBF in a concentration-dependentmanner.When themucosa was exposed to allyl isothiocyanate (10 mM) repeatedly, this response showed a marked desensitization. The increased GMBF response caused by allyl isothiocyanate was entirely blocked by co-application with HC-030031, but not BCTC. Interestingly, the increased GMBF in response to allyl isothiocyanate was significantly attenuated, but not completely disappear, by chemical deafferentation following systemic capsaicin injections (total dose: 100 mg/kg), although the increased GMBF in response to capsaicin was completely abolished by chemical deafferentation. The increased GMBF response to capsaicin was also significantly decreased in allyl isothiocyanate-desensitized preparation of the ex-vivo stomach. CONCLUSION: These results suggested that allyl isothiocyanate increases GMBF through activation of TRPA1, but not TRPV1. It is assumed that the increased GMBF in response to allyl isothiocyanate is mediated by TRPA1 located on both TRPV1-expresseing and TRPV1 nonexpressing sensory nerves.

Molecular Determinants of TRPV1 Stimulation by Mustard Oil

Mustard oil (MO, allyl isothiocyanate) is a highly electrophylic compound known to produce irritation, inflammation and pain. As such, it is used frequently in research and its effects were initially thought to be solely mediated by activation of the cation channel TRPA1. In contrast to this view, we have recently shown that MO also acts as an agonist of the capsaicin and heat receptor TRPV1, but the underlying molecular mechanisms remain unknown. We have found that MO effects on TRPV1 were rapidly reversible upon washout and were not significantly altered by the mutation of the residue C155, a residue which is essential for the action of the electrophile allicin. Hence, MO action on TRPV1 seems not to be mediated by long lasting covalent modification of cysteine residues. Notably, MO seems to act in a similar way as capsaicin, causing a shift of the voltage dependence of channel activation to more negative potentials. We therefore tested MO effects on TRPV1 mutants known to alter the sensitivity to capsaicin. We found that the substitution S512Y in the putative transmembrane domain 3 significantly reduces the ability of MO to induce stimulation and to shift of the voltage dependence of channel activation. In addition, we found that, similar to capsaicin, MO cross sensitize with other TRPV1 stimulators such as heat and extracellular protons. The molecular determinants of these interactions may help to understand the mechanisms of action of MO in real conditions (physiological temperatures and acidosis), as well as the mechanisms of MO-induced hyperalgesia to heat application.

The Capsaicin Receptor TRPV1 Is a Crucial Mediator of the Noxious Effects of Mustard Oil

Mustard oil (MO) is a plant-derived irritant that has been extensively used in experimental models to induce pain and inflammation. The noxious effects of MO are currently ascribed to specific activation of the cation channel TRPA1 in nociceptive neurons. In contrast to this view, we show here that the capsaicin receptor TRPV1 has a surprisingly large contribution to aversive and pain responses and visceral irritation induced by MO. Furthermore, we found that this can be explained by previously unknown properties of this compound. First, MO has a bimodal effect on TRPA1, producing current inhibition at millimolar concentrations. Second, it directly and stably activates mouse and human recombinant TRPV1, as well as TRPV1 channels in mouse sensory neurons. Finally, physiological temperatures enhance MO-induced TRPV1 stimulation. Our results refute the dogma that TRPA1 is the sole nocisensor for MO and motivate a revision of the putative roles of these channels in models of MO-induced pain and inflammation. We propose that TRPV1 has a generalized role in the detection of irritant botanical defensive traits and in the coevolution of multiple mammalian and plant species.

Drosophila TRPA1 Channel Is Required to Avoid the Naturally Occurring Insect Repellent Citronellal

Plants produce insect repellents, such as citronellal, which is the main component of citronellal oil. However, the molecular pathways through which insects sense botanical repellents are unknown. Here, we show that Drosophila use two pathways for direct avoidance of citronellal. The olfactory coreceptor OR83b contributes to citronellal repulsion and is essential for citronellal-evoked action potentials. Mutations affecting the Ca(2+)-permeable cation channel TRPA1 result in a comparable defect in avoiding citronellal vapor. The TRPA1-dependent aversion to citronellal relies on a G protein (Gq)/phospholipase C (PLC) signaling cascade rather than direct detection of citronellal by TRPA1. Loss of TRPA1, Gq, or PLC causes an increase in the frequency of citronellal-evoked action potentials in olfactory receptor neurons. Absence of the Ca(2+)-activated K(+) channel (BK channel) Slowpoke results in a similar impairment in citronellal avoidance and an increase in the frequency of action potentials. These results suggest that TRPA1 is required for activation of a BK channel to modulate citronellal-evoked action potentials and for aversion to citronellal. In contrast to Drosophila TRPA1, Anopheles gambiae TRPA1 is directly and potently activated by citronellal, thereby raising the possibility that mosquito TRPA1 may be a target for developing improved repellents to reduce insect-borne diseases such as malaria.

Agonist-Induced Changes in Ca2+ Permeation through the Nociceptor Cation Channel TRPA1

The Ca2+-permeable cation channel TRPA1 acts as an ionotropic receptor for various pungent compounds and as a noxious cold sensor in sensory neurons. It is unclear what proportion of the TRPA1-mediated current is carried by Ca2+ ions and how the permeation pathway changes during stimulation. Here, based on the relative permeability of the nonstimulated channel to cations of different size, we estimated a pore diameter of ∼11 Å. Combined patch-clamp and Fura-2 fluorescence recordings revealed that with 2 mM extracellular Ca2+, and at a membrane potential of −80 mV, ∼17% of the inward TRPA1 current is carried by Ca2+. Stimulation with mustard oil evoked an apparent dilatation of the pore of 3 Å and an increase in divalent cation selectivity and fractional Ca2+ current. Mutations in the putative pore that reduced the divalent permeability and fractional Ca2+ current also prevented mustard-oil-induced increases in Ca2+ permeation. It is interesting that fractional Ca2+ currents for wild-type and mutant TRPA1 were consistently higher than values predicted based on biionic reversal potentials using the Goldman-Hodgkin-Katz equation, suggesting that binding of Ca2+ in the pore hinders monovalent cation permeation. We conclude that the pore of TRPA1 is dynamic and supports a surprisingly large Ca2+ influx.

A Dietary Agonist of Transient Receptor Potential Cation Channel V3 Elicits Endothelium-Dependent Vasodilation

The Mediterranean diet may be responsible for lower cardiovascular disease rates in Southern versus Northern European countries. Oregano is used abundantly in Mediterranean cooking, but potential cardiovascular benefits have not been investigated. Carvacrol, present in oregano, activates the transient receptor potential (TRP) cation channels TRPA1 and TRPV3. We hypothesized that chemosensing of this dietary molecule by TRP channels in the endothelium promotes arterial relaxation. TRPA1 and TRPV3 were detected in the endothelium of intact arteries. Carvacrol causes concentration-dependent increases in the intracellular [Ca(2+)] of native cerebral artery endothelial cells and is more potent (EC(50) = 34 microM) than the TRPA1 agonist allyl isothiocyanate (EC(50) = 400 microM) or the TRPV3 agonist eugenol (EC(50) = 2.3 mM). Carvacrol also activates TRPV3-like cation currents in cerebral artery endothelial cells. Carvacrol elicits vasodilation of intact cerebral arteries (EC(50) = 4.1 microM) that is accompanied by smooth muscle hyperpolarization and a decrease in the intracellular [Ca(2+)] of arterial myocytes. Endothelium disruption inhibits carvacrol-induced vasodilation, but block of nitric-oxide synthase and cyclooxygenase activity does not alter the response. Vasodilation in response to carvacrol is inhibited when blockers of Ca(2+)-activated K(+) channels are present in the lumen or when the inwardly rectifying K(+) channel blocker BaCl(2) is present in the superfusion bath. Carvacrol-induced dilation is not diminished by a TRPA1 antagonist but is inhibited by the TRPV blocker ruthenium red. Our findings show that oregano can relax arteries by activating TRPV3 channels in the endothelium. This effect may account for some of the cardioprotective effects of the Mediterranean diet.

TRPA1 regulates gastrointestinal motility through serotonin release from enterochromaffin cells

Serotonin (5-hydroxytryptamine; 5-HT) is abundantly present throughout the gastrointestinal tract and stored mostly in enterochromaffin (EC) cells, which are located on the mucosal surface. 5-HT released from EC cells stimulate both intrinsic and extrinsic nerves, which results in various physiological and pathophysiological responses, such as gastrointestinal contractions. EC cells are believed to have the ability to respond to the chemical composition of the luminal contents of the gut; however, the underlying molecular and cellular mechanisms have not been identified. Here, we demonstrate that the transient receptor potential (TRP) cation channel TRPA1, which is activated by pungent compounds or cold temperature, is highly expressed in EC cells. We also found that TRPA1 agonists, including allyl isothiocyanate and cinnamaldehyde, stimulate EC cell functions, such as increasing intracellular Ca(2+) levels and 5-HT release, by using highly concentrated EC cell fractions and a model of EC cell function, the RIN14B cell line. Furthermore, we showed that allyl isothiocyanate promotes the contraction of isolated guinea pig ileum via the 5-HT(3) receptor. Taken together, our results indicate that TRPA1 acts as a sensor molecule for EC cells and may regulate gastrointestinal function.

Inhibition of TRPM8 by Icilin Distinct from Desensitization Induced by Menthol and Menthol Derivatives

TRPM8 is a cation channel activated by cold temperatures and the chemical stimuli menthol and icilin. Both compounds use different mechanisms of current activation; amino acid residues within the S2-S3 linker have been identified critical for current activation by icilin but not by menthol. Current decline in the course of menthol stimulation reflects Ca(2+)-dependent desensitization attributed to phosphatidylinositol 4,5-bisphosphate depletion. Carboxyamide derivatives chemically resembling menthol have been described as activators of TRPM8 analogous to icilin. Our aim was a detailed analysis of whether differences exist between all these substances with respect to their activation and inactivation of currents. We studied wild-type TRPM8 as well as an s3-TRPM8 mutant with mutations in the S2-S3 linker region that could not be activated by icilin. Menthol and menthol derivatives behaved indistinguishable in evoking currents through both channels in a Ca(2+)-independent manner as well as inducing Ca(2+)-dependent desensitization. Icilin, in contrast, activated currents only in wild type TRPM8 and in the presence of Ca(2+). Moreover, it completely reversed currents induced by menthol, menthol derivatives, and cold temperatures in wild type TRPM8 and s3-TRPM8; this current inhibition was independent of Ca(2+). Finally, icilin suppressed current activation by the other agonists. None of the inhibiting effects of icilin occurred in the cation channel TRPA1 that is also stimulated by both menthol and icilin. Thus, icilin specifically inhibits TRPM8 independently of its interaction site within the S2-S3 linker through a process distinct from desensitization.

Activation of TRPA1 by Farnesyl Thiosalicylic Acid

The nonselective cation channel TRPA1 (ANKTM1, p120) is a potential mediator of pain, and selective pharmacological modulation of this channel may be analgesic. Although several TRPA1 activators exist, these tend to be either reactive or of low potency and/or selectivity. The aim of the present study, therefore, was to identify novel TRPA1 agonists. Using a combination of calcium fluorescent assays and whole-cell electrophysiology, we discovered several compounds that possess potent, selective TRPA1-activating activity, including several lipid compounds (farnesyl thiosalicylic acid, farnesyl thioacetic acid, 15-deoxy-Delta(12,14)-prostaglandin J(2), and 5,8,11,14-eicosatetraynoic acid), and two marketed drugs: disulfiram (Antabuse; a compound used in the treatment of alcohol abuse) and the antifungal agent chlordantoin. Farnesyl thiosalicylic acid activates the channel in excised patches and in the absence of calcium. Furthermore, using a quadruple TRPA1 mutant, we show that the mechanism of action of farnesyl thiosalicylic acid differs from that of the reactive electrophilic reagent allylisothiocyanate. As a TRPA1 agonist with a potentially novel mechanism of action, farnesyl thiosalicylic acid may be useful in the study of TRPA1 channels.