The exceptionally high reactivity of Cys 621 is critical for electrophilic activation of the sensory nerve ion channel TRPA1

Activation of the sensory nerve ion channel TRPA1 by electrophiles is the key mechanism that initiates nociceptive signaling, and leads to defensive reflexes and avoidance behaviors, during oxidative stress in mammals. TRPA1 is rapidly activated by subtoxic levels of electrophiles, but it is unclear how TRPA1 outcompetes cellular antioxidants that protect cytosolic proteins from electrophiles. Here, using physiologically relevant exposures, we demonstrate that electrophiles react with cysteine residues on mammalian TRPA1 at rates that exceed the reactivity of typical cysteines by 6,000-fold and that also exceed the reactivity of antioxidant enzymes. We show that TRPA1 possesses a complex reactive cysteine profile in which C621 is necessary for electrophile-induced binding and activation. Modeling of deprotonation energies suggests that K620 contributes to C621 reactivity and mutation of K620 alone greatly reduces the effect of electrophiles on TRPA1. Nevertheless, binding of electrophiles to C621 is not sufficient for activation, which also depends on the function of another reactive cysteine (C665). Together, our results demonstrate that TRPA1 acts as an effective electrophilic sensor because of the exceptionally high reactivity of C621.     

Reactions of electrophiles with nucleophilic thiolate sites: relevance to pathophysiological mechanisms and remediation

Electrophiles are electron-deficient species that form covalent bonds with electron-rich nucleophiles. In biological systems, reversible electrophile–nucleophile interactions mediate basal cytophysiological functions (e.g. enzyme regulation through S-nitrosylation), whereas irreversible electrophilic adduction of cellular macromolecules is involved in pathogenic processes that underlie many disease and injury states. The nucleophiles most often targeted by electrophiles are side chains on protein amino acids (e.g. Cys, His, and Lys) and aromatic nitrogen sites on DNA bases (e.g. guanine N7). The sulfhydryl thiol (RSH) side chain of cysteine residues is a weak nucleophile that can be ionized in specific conditions to a more reactive nucleophilic thiolate (RS^?). This review will focus on electrophile interactions with cysteine thiolates and the pathophysiological consequences that result from irreversible electrophile modification of this anionic sulfur. According to the Hard and Soft, Acids and Bases (HSAB) theory of Pearson, electrophiles and nucleophiles can be classified as either soft or hard depending on their relative polarizability. HSAB theory suggests that electrophiles will preferentially and more rapidly form covalent adducts with nucleophiles of comparable softness or hardness. Application of HSAB principles, in conjunction with in vitro and proteomic studies, have indicated that soft electrophiles of broad chemical classes selectively form covalent Michael-type adducts with soft, highly reactive cysteine thiolate nucleophiles. Therefore, these electrophiles exhibit a common mechanism of cytotoxicity. As we will discuss, this level of detailed mechanistic understanding is a necessary prerequisite for the rational development of effective prevention and treatment strategies for electrophile-based pathogenic states.     

Identification of Significant Amino Acids in Multiple Transmembrane Domains of Human Transient Receptor Potential Ankyrin 1 (TRPA1) for Activation by Eudesmol,an Oxygenized Sesquiterpene in Hop Essential Oil

Transient receptor potential ankyrin 1 (TRPA1) is a calcium-permeable non-selective cation channel that is activated by various noxious or irritant substances in nature, including spicy compounds. Many TRPA1 chemical activators have been reported; however, only limited information is available regarding the amino acid residues that contribute to the activation by non-electrophilic activators, whereas activation mechanisms by electrophilic ligands have been well characterized. We used intracellular Ca²⁺ measurements and whole-cell patch clamp recordings to show that eudesmol, an oxygenated sesquiterpene present at high concentrations in the essential oil of hop cultivar Hallertau Hersbrucker, could activate human TRPA1. Gradual activation of inward currents with outward rectification by eudesmol was observed in human embryonic kidney-derived 293 cells expressing human TRPA1. This activation was completely blocked by a TRPA1-specific inhibitor, HC03–0031. We identified three critical amino acid residues in human TRPA1 in putative transmembrane domains 3, 4, and 5, namely threonine at 813, tyrosine at 840, and serine at 873, for activation by β-eudesmol in a systematic mutational study. Our results revealed a new TRPA1 activator in hop essential oil and provide a novel insight into mechanisms of human TRPA1 activation by non-electrophilic chemicals.     

Ultraviolet light and photosensitising agents activate TRPA1 via generation of oxidative stress

TRPA1, a Ca(2+)-permeable cation channel that is expressed in sensory neurones, is involved in the perception of chemical irritants and mechanical hyperalgesia. TRPA1 is activated by either covalent or reversible binding of various chemical compounds, including allylisothiocyanate or acrolein, and is further sensitised by increases in the intracellular Ca(2+) concentration. We here demonstrate that TRPA1 confers a sensitivity towards near ultraviolet (UVA) light, which rapidly causes Ca(2+) entry. In electrophysiological recordings in whole cell and inside out modes, exposure to UVA light activated typical TRPA1 currents in a wavelength-dependent and membrane-delimited manner. In the presence of the photosensitising agents acridine orange (100 nM) or hypericin (10 nM), the sensitivity of light-induced TRPA1 activation was increased and extended towards the visible spectrum. Since extracellular application of hydrogen peroxide mimicked the effect of UVA irradiation and since dithiothreitol partly reversed the activation by UVA exposure, we conclude that reactive oxygen species may mediate the light-induced activation of TRPA1. Accordingly, hydrogen peroxide induced a TRPA1 activation with a membrane-delimited mode of action that was attenuated by dithiothreitol. Intracellular but not extracellular application of FeSO(4), which catalyses the formation of highly reactive hydroxyl radicals potentiated the hydrogen peroxide-stimulated TRPA1 activation. We conclude that, via generation of reactive oxygen species, light-induced TRPA1 activation provides an additional mode of activation, which renders TRPA1 a likely molecular candidate in processes leading to painful or burning sensations during photodynamic therapy or upon local application of hydrogen peroxide.     

Molecular characterization of TRPA1 channel activation by cysteine-reactive inflammatory mediators

TRPA1 is a member of the transient receptor potential (TRP) cation channel family, and is predominantly expressed in nociceptive neurons of dorsal root ganglia (DRG) and trigeminal ganglia. Activation of TRPA1 by environmental irritants such as mustard oil, allicin, and acrolein causes acute pain. However, the endogenous ligands that directly activate TRPA1 remain elusive in inflammation. Here, we show that a variety of inflammatory mediators (15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2), nitric oxide (NO), hydrogen peroxide (H2O2), and proton (H+)) activate human TRPA1 heterologously expressed in HEK cells. These inflammatory mediators induced robust Ca2+ influx in a subset of mouse DRG neurons. The TRP channel blocker ruthenium red almost completely inhibited neuronal responses by 15d-PGJ2 and NO, but partially suppressed responses to H2O2 and H+. Functional characterization of site-directed cysteine mutants of TRPA1 in combination with labeling experiments using biotinylated 15d-PGJ2 demonstrated that modifications of cytoplasmic N-terminal cysteines (Cys421 and Cys621) were responsible for the activation of TRPA1 by 15d-PGJ2. In TRPA1 responses to other cysteine-reactive inflammatory mediators, such as NO and H2O2, the extents of impairment by respective cysteine mutations differed from those in TRPA1 responses to 15d-PGJ2. Interestingly, the Cys421 mutation critically impaired the TRPA1 response to H+ as well. Our findings suggest that TRPA1 channels are targeted by an array of inflammatory mediators to elicit inflammatory pain in the nervous system.     

C-terminal acidic cluster is involved in Ca2+-induced regulation of human transient receptor potential ankyrin 1 channel

The transient receptor potential ankyrin 1 (TRPA1) channel is a Ca(2+)-permeable cation channel whose activation results from a complex synergy between distinct activation sites, one of which is especially important for determining its sensitivity to chemical, voltage and cold stimuli. From the cytoplasmic side, TRPA1 is critically regulated by Ca(2+) ions, and this mechanism represents a self-modulating feedback loop that first augments and then inhibits the initial activation. We investigated the contribution of the cluster of acidic residues in the distal C terminus of TRPA1 in these processes using mutagenesis, whole cell electrophysiology, and molecular dynamics simulations and found that the neutralization of four conserved residues, namely Glu(1077) and Asp(1080)-Asp(1082) in human TRPA1, had strong effects on the Ca(2+)- and voltage-dependent potentiation and/or inactivation of agonist-induced responses. The surprising finding was that truncation of the C terminus by only 20 residues selectively slowed down the Ca(2+)-dependent inactivation 2.9-fold without affecting other functional parameters. Our findings identify the conserved acidic motif in the C terminus that is actively involved in TRPA1 regulation by Ca(2+).     

The Molecular Basis for Species-specific Activation of Human TRPA1 Protein by Protons Involves Poorly Conserved Residues within Transmembrane Domains 5 and 6

The surveillance of acid-base homeostasis is concerted by diverse mechanisms, including an activation of sensory afferents. Proton-evoked activation of rodent sensory neurons is mainly mediated by the capsaicin receptor TRPV1 and acid-sensing ion channels. In this study, we demonstrate that extracellular acidosis activates and sensitizes the human irritant receptor TRPA1 (hTRPA1). Proton-evoked membrane currents and calcium influx through hTRPA1 occurred at physiological acidic pH values, were concentration-dependent, and were blocked by the selective TRPA1 antagonist {"type":"entrez-nucleotide","attrs":{"text":"HC030031","term_id":"262060681","term_text":"HC030031"}}HC030031. Both rodent and rhesus monkey TRPA1 failed to respond to extracellular acidosis, and protons even inhibited rodent TRPA1. Accordingly, mouse dorsal root ganglion neurons lacking TRPV1 only responded to protons when hTRPA1 was expressed heterologously. This species-specific activation of hTRPA1 by protons was reversed in both mouse and rhesus monkey TRPA1 by exchange of distinct residues within transmembrane domains 5 and 6. Furthermore, protons seem to interact with an extracellular interaction site to gate TRPA1 and not via a modification of intracellular N-terminal cysteines known as important interaction sites for electrophilic TRPA1 agonists. Our data suggest that hTRPA1 acts as a sensor for extracellular acidosis in human sensory neurons and should thus be taken into account as a yet unrecognized transduction molecule for proton-evoked pain and inflammation. The species specificity of this property is unique among known endogenous TRPA1 agonists, possibly indicating that evolutionary pressure enforced TRPA1 to inherit the role as an acid sensor in human sensory neurons.     

A TRPA1-dependent mechanism for the pungent sensation of weak acids

Acetic acid produces an irritating sensation that can be attributed to activation of nociceptors within the trigeminal ganglion that innervate the nasal or oral cavities. These sensory neurons sense a diverse array of noxious agents in the environment, allowing animals to actively avoid tissue damage. Although receptor mechanisms have been identified for many noxious chemicals, the mechanisms by which animals detect weak acids, such as acetic acid, are less well understood. Weak acids are only partially dissociated at neutral pH and, as such, some can cross the cell membrane, acidifying the cell cytosol. The nociceptor ion channel TRPA1 is activated by CO(2), through gating of the channel by intracellular protons, making it a candidate to more generally mediate sensory responses to weak acids. To test this possibility, we measured responses to weak acids from heterologously expressed TRPA1 channels and trigeminal neurons with patch clamp recording and Ca(2+) microfluorometry. Our results show that heterologously expressed TRPA1 currents can be induced by a series of weak organic acids, including acetic, propionic, formic, and lactic acid, but not by strong acids. Notably, the degree of channel activation was predicted by the degree of intracellular acidification produced by each acid, suggesting that intracellular protons are the proximate stimulus that gates the channel. Responses to weak acids produced a Ca(2+)-independent inactivation that precluded further activation by weak acids or reactive chemicals, whereas preactivation by reactive electrophiles sensitized TRPA1 channels to weak acids. Importantly, responses of trigeminal neurons to weak acids were highly overrepresented in the subpopulation of TRPA1-expressing neurons and were severely reduced in neurons from TRPA1 knockout mice. We conclude that TRPA1 is a general sensor for weak acids that produce intracellular acidification and suggest that it functions within the pain pathway to mediate sensitivity to cellular acidosis.     

Voltage-dependent modulation of TRPA1 currents by diphenhydramine

Diphenhydramine (DPH) has been broadly used to treat allergy. When used as a topical medicine, DPH temporarily relieves itching and pain. Although transient receptor potential type A1 (TRPA1) channel is known to play roles in both acute and chronic itch and pain, whether DPH affects the activities of TRPA1 remains unclear. Using whole-cell patch clamp recordings, we demonstrated that DPH modulates the voltage-dependence of TRPA1. When co-applied with a TRPA1 agonist, DPH significantly enhanced the inward currents while suppressing the outward currents of TRPA1, converting the channel from outwardly rectifying to inwardly rectifying. This effect of DPH occurred no matter TRPA1 was activated by an electrophilic or non-electrophilic agonist and for both mouse and human TRPA1. The modulation of TRPA1 by DPH was maintained in the L906C mutant, which by itself also causes inward rectification of TRPA1, indicating that additional acting sites are present for the modulation of TRPA1 currents by DPH. Our recordings also revealed that DPH partially blocked capsaicin evoked TRPV1 currents. These data suggest that DPH may exert its therapeutic effects on itch and pain, through modulation of TRPA1 in a voltage-dependent fashion.     

Molecular characterization of TRPA1 channel activation by cysteine-reactive inflammatory mediators

TRPA1 is a member of the transient receptor potential (TRP) cation channel family, and is predominantly expressed in nociceptive neurons of dorsal root ganglia (DRG) and trigeminal ganglia. Activation of TRPA1 by environmental irritants such as mustard oil, allicin and acrolein causes acute pain. However, the endogenous ligands that directly activate TRPA1 remain elusive in inflammation. Here, we show that a variety of inflammatory mediators (15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)), nitric oxide (NO), hydrogen peroxide (H(2)O(2)), and proton (H(+))) activate human TRPA1 heterologously expressed in HEK cells. These inflammatory mediators induced robust Ca(2+) influx in a subset of mouse DRG neurons. The TRP channel blocker ruthenium red almost completely inhibited neuronal responses by 15d-PGJ(2) and NO, but partially suppressed responses to H(2)O(2) and H(+). Functional characterization of site-directed cysteine mutants of TRPA1 in combination with labeling experiments using biotinylated 15d-PGJ(2) demonstrated that modifications of cytoplasmic N-terminal cysteines (Cys421 and Cys621) were responsible for the activation of TRPA1 by 15d-PGJ(2). In TRPA1 responses to other cysteine-reactive inflammatory mediators, such as NO and H(2)O(2), the extent of impairment by respective cysteine mutations differed from those in TRPA1 responses to 15d-PGJ(2). Interestingly, the Cys421 mutation critically impaired the TRPA1 response to H(+) as well. Our findings suggest that TRPA1 channels are targeted by an array of inflammatory mediators to elicit inflammatory pain in the nervous system.     

Cell signalling by reactive lipid species: new concepts and molecular mechanisms

The process of lipid peroxidation is widespread in biology and is mediated through both enzymatic and non-enzymatic pathways. A significant proportion of the oxidized lipid products are electrophilic in nature, the RLS (reactive lipid species), and react with cellular nucleophiles such as the amino acids cysteine, lysine and histidine. Cell signalling by electrophiles appears to be limited to the modification of cysteine residues in proteins, whereas non-specific toxic effects involve modification of other nucleophiles. RLS have been found to participate in several physiological pathways including resolution of inflammation, cell death and induction of cellular antioxidants through the modification of specific signalling proteins. The covalent modification of proteins endows some unique features to this signalling mechanism which we have termed the 'covalent advantage'. For example, covalent modification of signalling proteins allows for the accumulation of a signal over time. The activation of cell signalling pathways by electrophiles is hierarchical and depends on a complex interaction of factors such as the intrinsic chemical reactivity of the electrophile, the intracellular domain to which it is exposed and steric factors. This introduces the concept of electrophilic signalling domains in which the production of the lipid electrophile is in close proximity to the thiol-containing signalling protein. In addition, we propose that the role of glutathione and associated enzymes is to insulate the signalling domain from uncontrolled electrophilic stress. The persistence of the signal is in turn regulated by the proteasomal pathway which may itself be subject to redox regulation by RLS. Cell death mediated by RLS is associated with bioenergetic dysfunction, and the damaged proteins are probably removed by the lysosome-autophagy pathway.     

Thermosensation and pain

We feel a wide range of temperatures spanning from cold to heat. Within this range, temperatures over about 43 degrees C and below about 15 degrees C evoke not only a thermal sensation, but also a feeling of pain. In mammals, six thermosensitive ion channels have been reported, all of which belong to the TRP (transient receptor potential) superfamily. These include TRPV1 (VR1), TRPV2 (VRL-1), TRPV3, TRPV4, TRPM8 (CMR1), and TRPA1 (ANKTM1). These channels exhibit distinct thermal activation thresholds (>43 degrees C for TRPV1, >52 degrees C for TRPV2, > approximately 34-38 degrees C for TRPV3, > approximately 27-35 degrees C for TRPV4, < approximately 25-28 degrees C for TRPM8 and <17 degrees C for TRPA1), and are expressed in primary sensory neurons as well as other tissues. The involvement of TRPV1 in thermal nociception has been demonstrated by multiple methods, including the analysis of TRPV1-deficient mice. TRPV2, TRPM8, and TRPA1 are also very likely to be involved in thermal nociception, because their activation thresholds are within the noxious range of temperatures.     

Inhibition of mitochondrial division through covalent modification of Drp1 protein by 15 deoxy-Δ-prostaglandin J2

Arachidonic acid derived endogenous electrophile 15d-PGJ2 has gained much attention in recent years due to its potent anti-proliferative and anti-inflammatory actions mediated through thiol modification of cysteine residues in its target proteins. Here, we show that 15d-PGJ2 at 1 μM concentration converts normal mitochondria into large elongated and interconnected mitochondria through direct binding to mitochondrial fission protein Drp1 and partial inhibition of its GTPase activity. Mitochondrial elongation induced by 15d-PGJ2 is accompanied by increased assembly of Drp1 into large oligomeric complexes through plausible intermolecular interactions. The role of decreased GTPase activity of Drp1 in the formation of large oligomeric complexes is evident when Drp1 is incubated with a non-cleavable GTP analog, GTPγS or by a mutation that inactivated GTPase activity of Drp1 (K38A). The mutation of cysteine residue (Cys644) in the GTPase effector domain, a reported target for modification by reactive electrophiles, to alanine mimicked K38A mutation induced Drp1 oligomerization and mitochondrial elongation, suggesting the importance of cysteine in GED to regulate the GTPase activity and mitochondrial morphology. Interestingly, treatment of K38A and C644A mutants with 15d-PGJ2 resulted in super oligomerization of both mutant Drp1s indicating that 15d-PGJ2 may further stabilize Drp1 oligomers formed by loss of GTPase activity through covalent modification of middle domain cysteine residues. The present study documents for the first time the regulation of a mitochondrial fission activity by a prostaglandin, which will provide clues for understanding the pathological and physiological consequences of accumulation of reactive electrophiles during oxidative stress, inflammation and degeneration.     

TRPV1 and TRPA1 Mediate Peripheral Nitric Oxide-Induced Nociception in Mice

Nitric oxide (NO) can induce acute pain in humans and plays an important role in pain sensitization caused by inflammation and injury in animal models. There is evidence that NO acts both in the central nervous system via a cyclic GMP pathway and in the periphery on sensory neurons through unknown mechanisms. It has recently been suggested that TRPV1 and TRPA1, two polymodal ion channels that sense noxious stimuli impinging on peripheral nociceptors, are activated by NO in heterologous systems. Here, we investigate the relevance of this activation. We demonstrate that NO donors directly activate TRPV1 and TRPA1 in isolated inside-out patch recordings. Cultured primary sensory neurons display both TRPV1- and TRPA1-dependent responses to NO donors. BH4, an essential co-factor for NO production, causes activation of a subset of DRG neurons as assayed by calcium imaging, and this activation is at least partly dependent on nitric oxide synthase activity. We show that BH4-induced calcium influx is ablated in DRG neurons from TRPA1/TRPV1 double knockout mice, suggesting that production of endogenous levels of NO can activate these ion channels. In behavioral assays, peripheral NO-induced nociception is compromised when TRPV1 and TRPA1 are both ablated. These results provide genetic evidence that the peripheral nociceptive action of NO is mediated by both TRPV1 and TRPA1.     

Peripheral non-viral MIDGE vector-driven delivery of β-endorphin in inflammatory pain

Background

TRPA1 has been implicated in both chemo- and mechanosensation. Recent work demonstrates that inhibiting TRPA1 function reduces mechanical hypersensitivity produced by inflammation. Furthermore, a broad range of chemical irritants require functional TRPA1 to exert their effects. In this study we use the ex-vivo skin-nerve preparation to directly determine the contribution of TRPA1 to mechanical- and chemical-evoked responses at the level of the primary afferent terminal.

Results

Acute application of HC-030031, a selective TRPA1 antagonist, inhibited all formalin responses in rat C fibers but had no effect on TRPV1 function, assessed by capsaicin responsiveness. Genetic ablation experiments corroborated the pharmacological findings as C fibers from wild type mice responded to both formalin and capsaicin, but fibers from their TRPA1-deficient littermates responded only to capsaicin. HC-030031 markedly reduced the mechanically-evoked action potential firing in rat and wild type mouse C fibers, particularly at high-intensity forces, but had no effect on the mechanical responsiveness of Aδ fiber nociceptors. Furthermore, HC-030031 had no effect on mechanically-evoked firing in C fibers from TRPA1-deficient mice, indicating that HC-030031 inhibits mechanically-evoked firing via a TRPA1-dependent mechanism.

Conclusion

Our data show that acute pharmacological blockade of TRPA1 at the cutaneous receptive field inhibits formalin-evoked activation and markedly reduces mechanically-evoked action potential firing in C fibers. Thus, functional TRPA1 at sensory afferent terminals in skin is required for their responsiveness to both noxious chemical and mechanical stimuli.     

Identification of proteins adducted by lipid peroxidation products in plasma and modifications of apolipoprotein A1 with a novel biotinylated phospholipid probe

Reactive electrophiles generated by lipid peroxidation are thought to contribute to cardiovascular disease and other oxidative stress-related pathologies by covalently modifying proteins and affecting critical protein functions. The difficulty of capturing and analyzing the relatively small fraction of modified proteins complicates identification of the protein targets of lipid electrophiles. We recently synthesized a biotin-modified linoleoylglycerylphosphatidylcholine probe called PLPBSO ( Tallman et al. Chem. Res. Toxicol. 2007, 20, 227-234 ), which forms typical linoleate oxidation products and covalent adducts with model peptides and proteins. Supplementation of human plasma with PLPBSO followed by free radical oxidation resulted in covalent adduction of PLPBSO to plasma proteins, which were isolated with streptavidin and identified by liquid chromatography-tandem mass spectrometry (LC-MS-MS). Among the most highly modified proteins was apolipoprotein A1 (ApoA1), which is the core component of high density lipoprotein (HDL). ApoA1 phospholipid adduct sites were mapped by LC-MS-MS of tryptic peptides following mild base hydrolysis to release esterified phospholipid adducts. Several carboxylated adducts formed from phospholipid-esterified 9,12-dioxo-10( E)-dodecenoic acid (KODA), 9-hydroxy, 12-oxo-10( E)-dodecenoic acid (HODA), 7-oxoheptanoic acid, 8-oxooctanoic acid, and 9-oxononanoic acid were identified. Free radical oxidations of isolated HDL also generated adducts with 4-hydroxynonenal (HNE) and other noncarboxylated electrophiles, but these were only sporadically identified in the PLPBSO-adducted ApoA1, suggesting a low stoichiometry of modification in the phospholipid-adducted protein. Both phospholipid electrophiles and HNE adducted His162, which resides in an ApoA1 domain involved in the activation of Lecithin-cholesterol acyltransferase and maturation of the HDL particle. ApoA1 lipid electrophile adducts may affect protein functions and provide useful biomarkers for oxidative stress.     

Differential cytotoxicity and intracellular calcium-signalling following activation of the calcium-permeable ion channels TRPV1 and TRPA1

Several members of the transient receptor channel (TRP) family can mediate a calcium-dependent cytotoxicity. In sensory neurons, vanilloids like capsaicin induce neurotoxicity by activating TRPV1. The closely related ion channel TRPA1 is also activated by irritants, but it is unclear if and how TRPA1 mediates cell death. In the present study we explored cytotoxicity and intracellular calcium signalling resulting from activation of TRPV1 and TRPA1, either heterologously expressed in HEK 293 cells or in native mouse dorsal root ganglion (DRG) neurons. While activation of TRPV1 by the vanilloids capsaicin, resiniferatoxin and anandamide results in calcium-dependent cell death, activation by protons and the oxidant chloramine-T failed to reduce cell viability. The TRPA1-agonists acrolein, carvacrol and capsazepine all induced cytotoxicity, but this effect is independent of TRPA1. Activation of both TRPA1 and TRPV1 triggers a strong influx of external calcium, but also a strong calcium-release from intracellular stores most likely including the endoplasmic reticulum (ER). Activation of TRPV1, but not TRPA1 also results in a strong increase of mitochondrial calcium both in HEK 293 cells and mouse DRG neurons. Our data demonstrate that activation of TRPV1, but not TRPA1 mediates a calcium-dependent cell death. While both receptors mediate a release of calcium from intracellular stores, only activation of TRPV1 seems to mediate a robust and probably lethal increase in mitochondrial calcium.     

Artepillin C,a Major Ingredient of Brazilian Propolis,Induces a Pungent Taste by Activating TRPA1 Channels

Brazilian green propolis is a popular health supplement because of its various biological properties. The ethanol extract of Brazilian green propolis (EEBP) is characteristic for its herb-like smell and unique pungent taste. However, the ingredients responsible for its pungency have not yet been identified. This study provides the first evidence that artepillin C is the main pungent ingredient in EEBP and that it potently activates human transient receptor potential ankyrin 1 (TRPA1) channels. EEBP was fractionated using column chromatography with a step gradient elution of an ethanol-water solution, and the fractions having the pungent taste were determined by sensory tests. HPLC analysis revealed that the pungent fraction was composed primarily of artepillin C, a prenylated derivative of cinnamic acid. Artepillin C was also identified as the pungent compound of EEBP by organoleptic examiners. Furthermore, the effects of artepillin C and other cinnamic acids found in EEBP on TRPA1 channels were examined by calcium imaging and plate reader-based assays in human TRPA1-expressing cells to investigate the molecular mechanisms underlying their pungent tastes. Artepillin C and baccharin activated the TRPA1 channel strongly, whereas drupanin caused a slight activation and p-coumaric acid showed no activation. Because the EC₅₀ values of artepillin C, baccharin, and allyl isothiocyanate were 1.8 µM, 15.5 µM, and 6.2 µM, respectively, artepillin C was more potent than the typical TRPA1 agonist allyl isothiocyanate. These findings strongly indicate that artepillin C is the main pungent ingredient in EEBP and stimulates a pungent taste by activating TRPA1 channels.