Oxidation of methionine residues activates the high-threshold heat-sensitive ion channel TRPV2.

Thermosensitive transient receptor potential (TRP) ion channels detect changes in ambient temperature to regulate body temperature and temperature-dependent cellular activity. Rodent orthologs of TRP vanilloid 2 (TRPV2) are activated by nonphysiological heat exceeding 50 °C, and human TRPV2 is heat-insensitive. TRPV2 is required for phagocytic activity of macrophages which are rarely exposed to excessive heat, but what activates TRPV2 in vivo remains elusive. Here we describe the molecular mechanism of an oxidation-induced temperature-dependent gating of TRPV2. While high concentrations of H2O2 induce a modest sensitization of heat-induced inward currents, the oxidant chloramine-T (ChT), ultraviolet A light, and photosensitizing agents producing reactive oxygen species (ROS) activate and sensitize TRPV2. This oxidation-induced activation also occurs in excised inside-out membrane patches, indicating a direct effect on TRPV2. The reducing agent dithiothreitol (DTT) in combination with methionine sulfoxide reductase partially reverses ChT-induced sensitization, and the substitution of the methionine (M) residues M528 and M607 to isoleucine almost abolishes oxidation-induced gating of rat TRPV2. Mass spectrometry on purified rat TRPV2 protein confirms oxidation of these residues. Finally, macrophages generate TRPV2-like heat-induced inward currents upon oxidation and exhibit reduced phagocytosis when exposed to the TRP channel inhibitor ruthenium red (RR) or to DTT. In summary, our data reveal a methionine-dependent redox sensitivity of TRPV2 which may be an important endogenous mechanism for regulation of TRPV2 activity and account for its pivotal role for phagocytosis in macrophages.

Distinct Mechanisms Account for In Vitro Activation and Sensitization of TRPV1 by the Porphyrin Hemin.

TRPV1 mediates pain occurring during sickling episodes in sickle cell disease (SCD). We examined if hemin, a porphyrin released during intravascular hemolysis modulates TRPV1. Calcium imaging and patch clamp were employed to examine effects of hemin on mouse dorsal root ganglion (DRG) neurons and HEK293t cells expressing TRPV1 and TRPA1. Hemin induced a concentration-dependent calcium influx in DRG neurons which was abolished by the unspecific TRP-channel inhibitor ruthenium red. The selective TRPV1-inhibitor BCTC or genetic deletion of TRPV1 only marginally impaired hemin-induced calcium influx in DRG neurons. While hTRPV1 expressed in HEK293 cells mediated a hemin-induced calcium influx which was blocked by BCTC, patch clamp recordings only showed potentiated proton- and heat-evoked currents. This effect was abolished by the PKC-inhibitor chelerythrine chloride and in protein kinase C (PKC)-insensitive TRPV1-mutants. Hemin-induced calcium influx through TRPV1 was only partly PKC-sensitive, but it was abolished by the reducing agent dithiothreitol (DTT). In contrast, hemin-induced potentiation of inward currents was not reduced by DTT. Hemin also induced a redox-dependent calcium influx, but not inward currents on hTRPA1. Our data suggest that hemin induces a PKC-mediated sensitization of TRPV1. However, it also acts as a photosensitizer when exposed to UVA-light used for calcium imaging. The resulting activation of redox-sensitive ion channels such as TRPV1 and TRPA1 may be an in vitro artifact with limited physiological relevance.

Quaternary Lidocaine Derivative QX-314 Activates and Permeates Human TRPV1 and TRPA1 to Produce Inhibition of Sodium Channels and Cytotoxicity.

BACKGROUND: The relatively membrane-impermeable lidocaine derivative QX-314 has been reported to permeate the ion channels transient receptor potential vanilloid 1 (TRPV1) and transient receptor potential cation channel, subfamily A, member 1 (TRPA1) to induce a selective inhibition of sensory neurons. This approach is effective in rodents, but it also seems to be associated with neurotoxicity. The authors examined whether the human isoforms of TRPV1 and TRPA1 allow intracellular entry of QX-314 to mediate sodium channel inhibition and cytotoxicity. METHODS: Human embryonic kidney 293 (HEK-293) cells expressing wild-type or mutant human (h) TRPV1 or TRPA1 constructs as well as the sodium channel Nav1.7 were investigated by means of patch clamp and ratiometric calcium imaging. Cytotoxicity was examined by flow cytometry. RESULTS: Activation of hTRPA1 by carvacrol and hTRPV1 by capsaicin produced a QX-314-independent reduction of sodium current amplitudes. However, permeation of QX-314 through hTRPV1 or hTRPA1 was evident by a concentration-dependent, use-dependent inhibition of Nav1.7 activated at 10 Hz. Five and 30 mM QX-314 activated hTRPV1 via mechanisms involving the intracellular vanilloid-binding domain and hTRPA1 via unknown mechanisms independent of intracellular cysteins. Expression of hTRPV1, but not hTRPA1, was associated with a QX-314-induced cytotoxicity (viable cells 48 ± 5% after 30 mM QX-314) that was ameliorated by the TRPV1 antagonist 4-(3-chloro-2-pyridinyl)-N-[4-(1,1-dimethylethyl)phenyl]-1-piperazinecarboxamide (viable cells 81 ± 5%). CONCLUSIONS: The study data demonstrate that QX-314 directly activates and permeates the human isoforms of TRPV1 and TRPA1 to induce inhibition of sodium channels, but also a TRPV1-dependent cytotoxicity. These results warrant further validation of this approach in more intact preparations and may be valuable for the development of this concept into clinical practice.

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 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.

Methylglyoxal activates nociceptors through transient receptor potential channel A1 (TRPA1): a possible mechanism of metabolic neuropathies.

Neuropathic pain can develop as an agonizing sequela of diabetes mellitus and chronic uremia. A chemical link between both conditions of altered metabolism is the highly reactive compound methylglyoxal (MG), which accumulates in all cells, in particular neurons, and leaks into plasma as an index of the severity of the disorder. The electrophilic structure of this cytotoxic ketoaldehyde suggests TRPA1, a receptor channel deeply involved in inflammatory and neuropathic pain, as a molecular target. We demonstrate that extracellularly applied MG accesses specific intracellular binding sites of TRPA1, activating inward currents and calcium influx in transfected cells and sensory neurons, slowing conduction velocity in unmyelinated peripheral nerve fibers, and stimulating release of proinflammatory neuropeptides from and action potential firing in cutaneous nociceptors. Using a model peptide of the N terminus of human TRPA1, we demonstrate the formation of disulfide bonds based on MG-induced modification of cysteines as a novel mechanism. In conclusion, MG is proposed to be a candidate metabolite that causes neuropathic pain in metabolic disorders and thus is a promising target for medicinal chemistry.

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

BACKGROUND AND PURPOSE: Itch is associated with several pathologies and is a common drug-induced side effect. Chloroquine (CQ) is 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. EXPERIMENTAL APPROACH: Patch clamp and calcium imaging were utilized to examine effects of CQ on TRPA1 and TRPV1 expressed in HEK 293T cells. KEY RESULTS: In calcium imaging, CQ induces a concentration-dependent but MrgprA3-independent activation of TRPA1 and TRPV1. Although CQ itself inhibits TRPA1 and TRPV1 in patch clamp recordings, co-application of CQ and ultraviolet A (UVA) light evokes membrane currents through both channels. This effect is inhibited by the reducing agent dithiothreitol (DTT) and is 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. CONCLUSION AND IMPLICATIONS: Our data reveal novel pharmacological properties of CQ, allowing activation of TRPA1 and TRPV1 via photosensitization as well as intracellular alkalosis. These findings add more complexity to the commonly accepted dogma that CQ-induced itch is specifically mediated by MrgprA3 coupling to TRPA1.

Gating of the capsaicin receptor TRPV1 by UVA-light and oxidants are mediated by distinct mechanisms.

Redox-sensitivity is a common property of several transient receptor potential (TRP) ion channels. Oxidants and UVA-light activate TRPV2 by oxidizing methionine pore residues which are conserved in the capsaicin-receptor TRPV1. However, the redox-sensitivity of TRPV1 is regarded to depend on intracellular cysteine residues. In this study we examined if TRPV1 is gated by UVA-light, and if the conserved methionine residues are relevant for redox-sensitivity of TRPV1. Patch clamp recordings were performed to explore wildtype (WT) and mutants of human TRPV1 (hTRPV1). UVA-light induced hTRPV1-mediated membrane currents and potentiated both proton- and heat-evoked currents. The reducing agent dithiothreitol (DTT) prevented and partially reversed UVA-light induced sensitization of hTRPV1. UVA-light induced sensitization was reduced in the mutant hTRPV1-C158A/C387S/C767S (hTRPV1-3C). The remaining sensitivity to UVA-light of hTRRPV1-3C was not further reduced upon exchange of the methionine residues M568 and M645. While UVA-induced sensitization was reduced in the protein kinase C-insensitive mutant hTRPV1-S502A/S801A, the PKC-inhibitors chelerythrine chloride, staurosporine and Gö6976 did not reduce UVA-induced effects on hTRPV1-WT. While hTRPV1-3C was insensitive to the cysteine-selective oxidant diamide, it displayed a residual sensitivity to H2O2 and chloramine-T. However, the exchange of M568 and M645 in hTRPV1-3C did not further reduce these effects. Our data demonstrate that oxidants and UVA-light gate hTRPV1 by cysteine-dependent as well as cysteine-independent mechanisms. In contrast to TRPV2, the methionine residues 568 and 645 seem to be of limited relevance for redox-sensitivity of hTRPV1. Finally, UVA-light induced gating of hTRPV1 does not seem to require activation of protein kinase C.

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.

Reactive metabolites of acetaminophen activate and sensitize the capsaicin receptor TRPV1.

The irritant receptor TRPA1 was suggested to mediate analgesic, antipyretic but also pro-inflammatory effects of the non-opioid analgesic acetaminophen, presumably due to channel activation by the reactive metabolites parabenzoquinone (pBQ) and N-acetyl-parabenzoquinonimine (NAPQI). Here we explored the effects of these metabolites on the capsaicin receptor TRPV1, another redox-sensitive ion channel expressed in sensory neurons. Both pBQ and NAPQI, but not acetaminophen irreversibly activated and sensitized recombinant human and rodent TRPV1 channels expressed in HEK 293 cells. The reducing agents dithiothreitol and N-acetylcysteine abolished these effects when co-applied with the metabolites, and both pBQ and NAPQI failed to gate TRPV1 following substitution of the intracellular cysteines 158, 391 and 767. NAPQI evoked a TRPV1-dependent increase in intracellular calcium and a potentiation of heat-evoked currents in mouse spinal sensory neurons. Although TRPV1 is expressed in mouse hepatocytes, inhibition of TRPV1 did not alleviate acetaminophen-induced hepatotoxicity. Finally, intracutaneously applied NAPQI evoked burning pain and neurogenic inflammation in human volunteers. Our data demonstrate that pBQ and NAQPI activate and sensitize TRPV1 by interacting with intracellular cysteines. While TRPV1 does not seem to mediate acetaminophen-induced hepatotoxicity, our data identify TRPV1 as a target of acetaminophen with a potential relevance for acetaminophen-induced analgesia, antipyresia and inflammation.