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

Abstract

2, 4-dinitrofluorobenzene (DNFB) and 2, 4-dinitrochlorobenzene (DNCB) are well known as skin sensitizers that can cause dermatitis. DNFB has shown to more potently sensitize skin; however, how DNFB and DNCB cause skin inflammation at a molecular level and why this difference in their sensitization ability is observed remain unknown. In this study, we aimed to identify the molecular targets and mechanisms on which DNFB and DNCB act. We used a fluorescent calcium imaging plate reader in an initial screening assay before patch-clamp recordings for validation. Molecular docking in combination with site-directed mutagenesis was then carried out to investigate DNFB and DNCB binding sites in the TRPA1 ion channel that may be selectively activated by these tow sensitizers. We found that DNFB and DNCB selectively activated TRPA1 channel with EC50 values of 2.3 ± 0.7 μM and 42.4 ± 20.9 μM, respectively. Single-channel recordings revealed that DNFB and DNCB increase the probability of channel opening and act on three residues (C621, E625, and Y658) critical for TRPA1 activation. Our findings may not only help explain the molecular mechanism underlying the dermatitis and pruritus caused by chemicals such as DNFB and DNCB, but also provide a molecular tool 7.5-fold more potent than the current TRPA1 activator allyl isothiocyanate (AITC) used for investigating TRPA1 channel pharmacology and pathology. 2, 4-dinitrofluorobenzene (DNFB) and 2, 4-dinitrochlorobenzene (DNCB) are well known as skin sensitizers that can cause dermatitis. DNFB has shown to more potently sensitize skin; however, how DNFB and DNCB cause skin inflammation at a molecular level and why this difference in their sensitization ability is observed remain unknown. In this study, we aimed to identify the molecular targets and mechanisms on which DNFB and DNCB act. We used a fluorescent calcium imaging plate reader in an initial screening assay before patch-clamp recordings for validation. Molecular docking in combination with site-directed mutagenesis was then carried out to investigate DNFB and DNCB binding sites in the TRPA1 ion channel that may be selectively activated by these tow sensitizers. We found that DNFB and DNCB selectively activated TRPA1 channel with EC50 values of 2.3 ± 0.7 μM and 42.4 ± 20.9 μM, respectively. Single-channel recordings revealed that DNFB and DNCB increase the probability of channel opening and act on three residues (C621, E625, and Y658) critical for TRPA1 activation. Our findings may not only help explain the molecular mechanism underlying the dermatitis and pruritus caused by chemicals such as DNFB and DNCB, but also provide a molecular tool 7.5-fold more potent than the current TRPA1 activator allyl isothiocyanate (AITC) used for investigating TRPA1 channel pharmacology and pathology. Nitrobenzene compounds such as 2, 4-dinitrofluorobenzene (DNFB) and 2, 4-dinitrochlorobenzene (DNCB) have long been known to cause skin irritation and sensitization, and they are used for establishment of skin inflammatory models in rodents (1Gouin O. L'Herondelle K. Lebonvallet N. Le Gall-Ianotto C. Sakka M. Buhé V. Plée-Gautier E. Carré J.-L. Lefeuvre L. Misery L. Le Garrec R. TRPV1 and TRPA1 in cutaneous neurogenic and chronic inflammation: Pro-inflammatory response induced by their activation and their sensitization.Protein Cell. 2017; 8: 644-661Google Scholar, 2Bourane S. Duan B. Koch S.C. Dalet A. Britz O. Garcia-Campmany L. Kim E. Cheng L.Z. Ghosh A. Ma Q.F. Goulding M. Gate control of mechanical itch by a subpopulation of spinal cord interneurons.Science. 2015; 350: 550-554Google Scholar, 3Barry D.M. Munanairi A. Chen Z.-F. Spinal mechanisms of itch transmission.Neurosci. Bull. 2017; 34: 156-164Google Scholar). Topical applications of DNFB and DNCB can mediate contact hypersensitivity and induce allergic contact dermatitis (ACD) (4Hsieh G.C. Kolano R.M. Andrews W. Fey T.A. Collins K.A. Gauvin D. Kawai M. Mollison K.W. Luly J.R. Immunosuppressant effects on improved Guinea pig contact hypersensitivity (CH) model: Induction with DNFB and elicitation with DNCB.FASEB J. 1996; 10: 1284Google Scholar, 5Garcia-Perez A. Occupational dermatitis from DNFB with cross sensitivity to DNCB.Contact Dermatitis. 1978; 4: 125-127Google Scholar). DNFB, commonly called Sanger’s reagent used for protein sequencing, also causes colitis in mice (6Rijnierse A. van Zijl K.M.F. Koster A.S. Nijkamp F.P. Kraneveld A.D. Beneficial effect of tachykinin NK1 receptor antagonism in the development of hapten-induced colitis in mice.Eur. J. Pharmacol. 2006; 548: 150-157Google Scholar, 7Rijnierse A. Koster A.S. Nijkamp F.P. Kraneveld A.D. TNF-alpha is crucial for the development of mast cell-dependent colitis in mice.Am. J. Physiol. Gastrointest. Liver Physiol. 2006; 291: G969-G976Google Scholar). Chemical DNFB as an allergen additionally elicits immune reactions by inducing mast cell degranulation and releases of histamine (3Barry D.M. Munanairi A. Chen Z.-F. Spinal mechanisms of itch transmission.Neurosci. Bull. 2017; 34: 156-164Google Scholar), interleukin-1 (IL-1) and prostaglandin E2 (PGE2) (8Parenti A. De Logu F. Geppetti P. Benemei S. What is the evidence for the role of TRP channels in inflammatory and immune cells?.Br. J. Pharmacol. 2016; 173: 953-969Google Scholar). It is of interest that both DNFB and DNCB share similar structures, but the skin is more sensitive to DNFB-induced irritation than DNCB (9Tingle M.D. Clarke J.B. Kitteringham N.R. Park B.K. Influence of glutathione conjugation on the immunogenicity of dinitrophenyl derivatives in the rat.Int. Arch. Allergy Appl. Immunol. 1990; 91: 160-165Google Scholar, 10Landsteiner K. Chase M.W. Studies on the sensitization of animals with simple chemical compounds: IX. Skin sensitization induced by injection of conjugates.J. Exp. Med. 1941; 73: 431-438Google Scholar). TRPA1 is a temperature-sensitive and calcium-permeable cation channel with a fourfold symmetry around the central ion conductance pathway and proximal cytoplasmic regions involved in electrophile detection (11Caterina M.J. TRP channel cannabinoid receptors in skin sensation, homeostasis, and inflammation.ACS Chem. Neurosci. 2014; 5: 1107-1116Google Scholar, 12Rodriguez A.L. Grier M.D. Jones C.K. Herman E.J. Kane A.S. Smith R.L. Williams R. Zhou Y. Marlo J.E. Days E.L. Blatt T.N. Jadhav S. Menon U.N. Vinson P.N. Rook J.M. et al.Discovery of novel allosteric modulators of metabotropic glutamate receptor subtype 5 reveals chemical and functional diversity and in vivo activity in rat behavioral models of anxiolytic and antipsychotic activity.Mol. Pharmacol. 2010; 78: 1105-1123Google Scholar, 13Lin J. Xu R. Hu L. You J. Jiang N. Li C. Che C. Wang Q. Xu Q. Li J. Zhao G. Interleukin-32 induced thymic stromal lymphopoietin plays a critical role in the inflammatory response in human corneal epithelium.Cell Signal. 2018; 49: 39-45Google Scholar). TRPA1 serves as a sensor for chemical irritants, such as mustard oil (MO) (14Gao Z.-R. Chen W.-Z. Liu M.-Z. Chen X.-J. Wan L. Zhang X.-Y. Yuan L. Lin J.-K. Wang M. Zhou L. Xu X.-H. Sun Y.-G. Tac1-expressing neurons in the periaqueductal gray facilitate the itch-scratching cycle via descending regulation.Neuron. 2019; 101: 45-59.e9Google Scholar), acrolein, cinnamaldehyde (CA) (15Ishibashi T. Takumida M. Akagi N. Hirakawa K. Anniko M. Expression of transient receptor potential vanilloid (TRPV) 1, 2, 3, and 4 in mouse inner ear.Acta Otolaryngol. 2008; 128: 1286-1293Google Scholar) and allyl isothiocyanate (AITC) (16Sandor Z. Dekany A. Kelemen D. Bencsik T. Papp R. Bartho L. The TRPA1 activator allyl isothiocyanate (AITC) contracts human jejunal muscle: Pharmacological analysis.Basic Clin. Pharmacol. Toxicol. 2016; 119: 341-342Google Scholar). TRPA1 is also activated by harmful electrophiles that are recognized by the channel via covalent modifications of specific cysteine residues located in the cytoplasmic C domain (17Sun Y.G. Chen Z.F. A gastrin-releasing peptide receptor mediates the itch sensation in the spinal cord.Nature. 2007; 448: 700-703Google Scholar). Nitrobenzene compound 2, 4, 6-trinitrobenzene sulfonic acid (TNBS) causes colitis by activating TRPA1 (18Engel M.A. Leffler A. Niedermirtl F. Babes A. Zimmermann K. Filipovic M.R. Izydorczyk I. Eberhardt M. Kichko T.I. Mueller-Tribbensee S.M. Khalil M. Siklosi N. Nau C. Ivanovic-Burmazovic I. Neuhuber W.L. et al.TRPA1 and substance P mediate colitis in mice.Gastroenterology. 2011; 141: 1346-1358Google Scholar). A growing number of evidences indicate that activation of transient receptor potential ankryn 1 (TRPA1) ion channels is involved in skin inflammation (19Tóth B.I. Oláh A. Szöllősi A.G. Bíró T. TRP channels in the skin.Br. J. Pharmacol. 2014; 171: 2568-2581Google Scholar). TRPA1 is robustly expressed in primary sensory nerve terminals (20Mandadi S. Roufogalis B.D. ThermoTRP channels in nociceptors: Taking a lead from capsaicin receptor TRPV1.Curr. Neuropharmacol. 2008; 6: 21-38Google Scholar) and numerous nonneuronal cell types of the skin (21Atoyan R. Shander D. Botchkareva N.V. Non-neuronal expression of transient receptor potential type A1 (TRPA1) in human skin.J. Invest. Dermatol. 2009; 129: 2312-2315Google Scholar) and CD4+ T lymphocytes that play a central role in the adaptive immune response (22Bertin S. Aoki-Nonaka Y. Lee J. de Jong P.R. Kim P. Han T. Yu T. To K. Takahashi N. Boland B.S. Chang J.T. Ho S.B. Herdman S. Corr M. Franco A. et al.The TRPA1 ion channel is expressed in CD4+T cells and restrains T-cell-mediated colitis through inhibition of TRPV1.Gut. 2017; 66: 1584-1596Google Scholar, 23Li M.W. Fan X.S. Yue Q.F. Hu F.Y. Zhang Y.M. Zhu C. The neuro-immune interaction in airway inflammation through TRPA1 expression in CD4+T cells of asthmatic mice.Int. Immunopharm. 2020; 86: 106696Google Scholar). Activation of TRPA1 by icilin in keratinocytes leads to an elevation of proinflammatory cytokine interleukin-1 which a role of TRPA1 in cutaneous inflammation (21Atoyan R. Shander D. Botchkareva N.V. Non-neuronal expression of transient receptor potential type A1 (TRPA1) in human skin.J. Invest. Dermatol. 2009; 129: 2312-2315Google Scholar). Pharmacological inhibition of TRPA1 inflammation of dermatitis J. Zhu Z. T. pruritus in chronic Immunol. Scholar, D. Chen C. Zhou W. Ma Y. Zhou W. F. TRPA1 inflammation of dermatitis by Scholar). specific activation of TRPA1 by leads to which is by in mice (18Engel M.A. Leffler A. Niedermirtl F. Babes A. Zimmermann K. Filipovic M.R. Izydorczyk I. Eberhardt M. Kichko T.I. Mueller-Tribbensee S.M. Khalil M. Siklosi N. Nau C. Ivanovic-Burmazovic I. Neuhuber W.L. et al.TRPA1 and substance P mediate colitis in mice.Gastroenterology. 2011; 141: 1346-1358Google Scholar). DNFB DNCB response in cells TRPA1 C. W. cells to by the activation of calcium 2016; Scholar, M. T. K. A. is a potent human TRPA1 2014; Scholar). compounds have been shown to cause calcium through TRPA1 activation on TRPA1 with at a molecular level unknown. In this study, we that chemicals DNFB and DNCB TRPA1 channels through binding to three residues critical for electrophile in the channel domain patch-clamp site-directed and molecular Our findings not only help explain the compounds contact skin irritation but also provide a molecular tool for of TRPA1 channel pharmacology in To the effect of DNFB and DNCB on TRPA1 and we the calcium fluorescent imaging of cells TRP channels in reader of DNFB and DNCB caused a increase of as with TRPA1 which was used as a control In was a of from TRPV1 and channels in response to DNFB and DNCB in the of that DNFB and DNCB selectively TRPA1 the of the TRP channel To the activation of TRPA1 channels by DNFB, we the of and channels expressed in cells in the of 5 μM DNFB and μM shown in 2, and of TRPA1 by the TRPA1 at μM In TRPV1 and channels not to DNFB and DNCB these channels activated by their such as 1 μM capsaicin for μM for and μM for with the from calcium fluorescent these that DNFB and DNCB are of To the of DNFB and DNCB on TRPA1 we the recordings of TRPA1 in the of of DNFB DNCB and observed a activation of TRPA1 with EC50 values of 2.3 ± 0.7 μM (DNFB) 3, A and and 42.4 ± 20.9 μM (DNCB) 3, and a TRPA1 also a activation of the channel current with an EC50 of ± μM 3, C and which is with a A. I. M. Y. N. A. S. of on in mice with Dermatol. 2006; Scholar). indicate that DNFB TRPA1 in with 7.5-fold than To DNFB and DNCB on TRPA1 we the recordings in an a of TRPA1 μM the channel opening from level 1 to level with the channel conductance ± and also the channel probability to ± from ± of control 4, DNFB at μM in a increase of the channel opening from level 1 to level 4 with channel conductance ± and channel probability to ± from ± 4, DNCB at μM in a increase of the channel opening from level 1 to level 4 with channel conductance ± and channel probability to ± from ± 4, a of the channel probability to ± from ± 4, A and DNFB caused than DNCB that DNFB TRPA1 by on channels through increase of channel To identify residues critical for DNFB and DNCB binding to we carried out the molecular docking of DNFB and DNCB the of human TRPA1 the shown in A and the docking reveals that DNFB and DNCB are the and in the domain that is involved in electrophile (17Sun Y.G. Chen Z.F. A gastrin-releasing peptide receptor mediates the itch sensation in the spinal cord.Nature. 2007; 448: 700-703Google Scholar). The of DNFB and DNCB are both through to at the of and in the and C and In in the binding of DNFB, the in DNFB with through the of to which causes the the and the interaction with and the interaction with to the and of the In the binding of DNCB, the of leads to the of these that help to the are also such as with DNFB through van which to be for the binding of DNFB to To that residues and in are for the DNFB and DNCB we of and shown in of μM DNFB μM DNCB was to of and and and In DNFB at μM the μM DNFB not the current of that is also for activation of TRPA1 by DNFB shown in μM DNFB not but DNCB at μM was to The that with is for DNFB but not DNCB that with difference may explain the in sensitization ability DNFB and that the three residues E625, and are critical for DNFB and DNCB binding to the TRPA1 channel domain and electrophile In this study, we TRPA1 as a molecular for chemicals DNFB and DNCB that can selectively the DNFB is more potent than that is of the commonly used of the DNFB and DNCB can increase the channel probability through binding to three residues E625, and in the channel domain that to electrophile (17Sun Y.G. Chen Z.F. A gastrin-releasing peptide receptor mediates the itch sensation in the spinal cord.Nature. 2007; 448: 700-703Google Scholar). Our of DNFB and DNCB as TRPA1 not only a tool for of the channel pharmacology and but also how compounds TRPA1 that is a potential for allergic contact dermatitis J. Zhu Z. T. pruritus in chronic Immunol. Scholar). DNFB has long been to cause skin sensitization that is on immune activation immune in the of allergic contact Immunol. Scholar), the underlying molecular mechanism DNFB as an allergen to in the which and inflammation (1Gouin O. L'Herondelle K. Lebonvallet N. Le Gall-Ianotto C. Sakka M. Buhé V. Plée-Gautier E. Carré J.-L. Lefeuvre L. Misery L. Le Garrec R. TRPV1 and TRPA1 in cutaneous neurogenic and chronic inflammation: Pro-inflammatory response induced by their activation and their sensitization.Protein Cell. 2017; 8: 644-661Google Scholar, 3Barry D.M. Munanairi A. Chen Z.-F. Spinal mechanisms of itch transmission.Neurosci. Bull. 2017; 34: 156-164Google Scholar). It is of interest to that DNFB-induced allergic response is of inflammatory response on the of DNFB that the inflammatory response E. M. P. A. N. A. A. J. J. M. hapten-induced colitis in mice not be a of inflammatory 2011; Scholar, A. Koster A.S. Nijkamp F.P. Kraneveld A.D. role for mast cells in the of hypersensitivity Immunol. 2006; Scholar). that DNFB and DNCB may also cause skin inflammation through and be a specific for DNFB-induced skin sensitization and TRPA1 as a sensor of chemical and of allergen can cause types of TRPA1 is expressed in sensory nerve in the skin and also cutaneous mast and and is involved in chronic pruritus (1Gouin O. L'Herondelle K. Lebonvallet N. Le Gall-Ianotto C. Sakka M. Buhé V. Plée-Gautier E. Carré J.-L. Lefeuvre L. Misery L. Le Garrec R. TRPV1 and TRPA1 in cutaneous neurogenic and chronic inflammation: Pro-inflammatory response induced by their activation and their sensitization.Protein Cell. 2017; 8: 644-661Google Scholar, L. T. D. D.M. D.M. The ion channel TRPA1 is for chronic Neurosci. Scholar). TRPA1 is activated by inflammatory from nonneuronal cells and is also for the of inflammatory and neurogenic which serves as a and of inflammatory D.M. M. M. A for Physiol. Scholar). such as and can TRPA1 neurons and inducing airway F. F. Geppetti P. F. A. S. M. R. in inflammatory from human J. 2007; Scholar, E. B. S. M. S. D. C. N. R. M. Geppetti P. R. neurogenic inflammation is by and the TRPA1 receptor in Clin. Invest. 2008; Scholar). In has been that skin inflammation induced by in mice can be improved by TRPA1 and D. Chen C. Zhou W. Ma Y. Zhou W. F. TRPA1 inflammation of dermatitis by Scholar, M. T. K. A. is a potent human TRPA1 2014; Scholar), which that can cause dermatitis by activating TRPA1 in have shown that TRP such as and are in skin and skin inflammation (19Tóth B.I. Oláh A. Szöllősi A.G. Bíró T. TRP channels in the skin.Br. J. Pharmacol. 2014; 171: 2568-2581Google Scholar, B. G. T. receptor potential cation channels in 2007; Scholar, L. C. The ion channel and J. Pharmacol. Scholar, J. J. Yu G. P. M.R. J. Yu W. A. Zhang Y. Liu S. S. Xu A. Cheng J. Liu Q. et receptor potential vanilloid and keratinocytes to allergic and chronic Allergy Clin. Immunol. 2018; 141: Scholar, M.D. Li T. Y. J.B. Activation of vanilloid of proinflammatory in human Pharmacol. Exp. Scholar, T. K. M. M. I. T. T. T. A. of the in on the development of allergic and dermatitis in Invest. Dermatol. 2009; 129: Scholar). Our indicate that DNFB and DNCB TRPA1 that is in a located in the channel domain (17Sun Y.G. Chen Z.F. A gastrin-releasing peptide receptor mediates the itch sensation in the spinal cord.Nature. 2007; 448: 700-703Google Scholar). The is in such as and (17Sun Y.G. Chen Z.F. A gastrin-releasing peptide receptor mediates the itch sensation in the spinal cord.Nature. 2007; 448: 700-703Google Scholar, A. D.M. D. TRP channel activation by covalent S. A. 2006; Scholar, S. D.M. Liu Munanairi A. Cheng W. P. Wan L. Liu S.B. K. Zhao N. J. et of by TRPV1 is for itch in sensory Signal. 2016; Scholar, S.M. Han S. Lee Y.M. Park Lee A peptide TRPV1 activation skin Dermatol. 2017; Scholar), and the electrophile is by a number of for the of electrophiles (17Sun Y.G. Chen Z.F. A gastrin-releasing peptide receptor mediates the itch sensation in the spinal cord.Nature. 2007; 448: 700-703Google Scholar). In the of TRPV1 F. M. E. R. K. S. Y. C. effects of on skin inflammatory and Invest. Dermatol. 2014; Scholar), Yu Y. Li M. in mice via potent Arch. Allergy Immunol. 2018; Scholar) and N. G. M. Yuan P. and of ion and 2018; Scholar) that these three channels the electrophile domain that serves to A.L. Grier M.D. Jones C.K. Herman E.J. Kane A.S. Smith R.L. Williams R. Zhou Y. Marlo J.E. Days E.L. Blatt T.N. Jadhav S. Menon U.N. Vinson P.N. Rook J.M. et al.Discovery of novel allosteric modulators of metabotropic glutamate receptor subtype 5 reveals chemical and functional diversity and in vivo activity in rat behavioral models of anxiolytic and antipsychotic activity.Mol. Pharmacol. 2010; 78: 1105-1123Google Scholar), which can explain the activation of TRPA1 by chemicals DNFB and DNCB M. T. K. A. is a potent human TRPA1 2014; Scholar). DNFB and DNCB as to TRPA1 through with and residues and a the of DNFB and with the that a binding a of TRPA1 channels D. S. P. V. K. D. K. D. Skin Exp. Med. 2017; Scholar). The binding DNFB and DNCB that TRPA1 the channel are used for the establishment of dermatitis immune in the of allergic contact Immunol. Scholar), which is that to and TRPA1 channel D. S. P. V. K. D. K. D. Skin Exp. Med. 2017; Scholar, Le from the of and Scholar). TRPA1 such as peptide can TRPA1 in a inducing channel and cause D. S. P. V. K. D. K. D. Skin Exp. Med. 2017; Scholar, D. Zhang J. K.A. L. T. S. E. de M. M.A. M. C. D. et of the J. 8: Scholar). are to as and are to their for to more activation of TRPA1 D. S. P. V. K. D. K. D. Skin Exp. Med. 2017; Scholar). In covalent are and are not in a of D. S. P. V. K. D. K. D. Skin Exp. Med. 2017; Scholar). TRPA1 such as and binding to the cysteine residues of the domain of can TRPA1 and and only cause (17Sun Y.G. Chen Z.F. A gastrin-releasing peptide receptor mediates the itch sensation in the spinal cord.Nature. 2007; 448: 700-703Google Scholar, S. M. M. N. A human Neurosci. 2019; Scholar, Y. reveals a mechanism of Pharmacol. Scholar). We that may cause more and channel than caused by which is with the for the channel caused by We also an in docking DNCB, the of DNFB, the a similar binding through the to DNCB, however, is potent that DNFB, that skin sensitization is than DNFB (1Gouin O. L'Herondelle K. Lebonvallet N. Le Gall-Ianotto C. Sakka M. Buhé V. Plée-Gautier E. Carré J.-L. Lefeuvre L. Misery L. Le Garrec R. TRPV1 and TRPA1 in cutaneous neurogenic and chronic inflammation: Pro-inflammatory response induced by their activation and their sensitization.Protein Cell. 2017; 8: 644-661Google Scholar, G.C. Kolano R.M. Andrews W. Fey T.A. Collins K.A. Gauvin D. Kawai M. Mollison K.W. Luly J.R. Immunosuppressant effects on improved Guinea pig contact hypersensitivity (CH) model: Induction with DNFB and elicitation with DNCB.FASEB J. 1996; 10: 1284Google Scholar). In the skin sensitizers DNFB and DNCB found to selectively TRPA1 channels through binding to the channel DNFB can as a molecular tool for of TRPA1 pharmacology and pathology. In inhibition of TRPA1 channel may a for of dermatitis. DNFB DNCB capsaicin and allyl isothiocyanate (AITC) from was as in before compounds as in before used for the of fluorescent calcium in the in for patch-clamp The human cells in with of of at with cells in a plate for calcium patch-clamp cells on The cells in a a well on a plate with of human TRPA1 and TRP The cells used to TRPA1 by The was used to the of calcium in a of cells with in assay cells with of TRP channels before at a of cells well in a plate for an at with with the calcium fluorescent in the at for and the values of by the at and with an of N. Sun Wang K. of a calcium fluorescent assay in for the screening of 2017; Scholar). patch-clamp recordings in of and recordings to an by at The a with the of to 5 5 to with a (1Gouin O. L'Herondelle K. Lebonvallet N. Le Gall-Ianotto C. Sakka M. Buhé V. Plée-Gautier E. Carré J.-L. Lefeuvre L. Misery L. Le Garrec R. TRPV1 and TRPA1 in cutaneous neurogenic and chronic inflammation: Pro-inflammatory response induced by their activation and their sensitization.Protein Cell. 2017; 8: 644-661Google 1 with for S. Duan B. Koch S.C. Dalet A. Britz O. Garcia-Campmany L. Kim E. Cheng L.Z. Ghosh A. Ma Q.F. Goulding M. Gate control of mechanical itch by a subpopulation of spinal cord interneurons.Science. 2015; 350: 550-554Google with for was to at a potential from to for with a potential of The of as the docking The binding to the TRPA1 binding (17Sun Y.G. Chen Z.F. A gastrin-releasing peptide receptor mediates the itch sensation in the spinal cord.Nature. 2007; 448: 700-703Google Scholar) and the The was to the with of DNFB and DNCB with as binding with The was used to site-directed in TRPA1 by to the of are expressed with the ± was by with and A of is to be are the The that they have of interest with the of this We are to Fan and Zhang for and in the molecular was by of and and the of and of S. and K. W. W. Y. and S. K. W. W. W. and S. K. W. W. S. and K. W. W. K. W. W. W. W. Y. and K. W. W. K. W. W. and

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