Molecular determinants for the chemical activation of the warmth-sensitive TRPV3 channel by the natural monoterpenoid carvacrol

Abstract

Transient receptor potential vanilloid 3 (TRPV3), robustly expressed in the skin, is a nonselective calcium-permeable cation channel activated by warm temperature, voltage, and certain chemicals. Natural monoterpenoid carvacrol from plant oregano is a known skin sensitizer or allergen that specifically activates TRPV3 channel. However, how carvacrol activates TRPV3 mechanistically remains to be understood. Here, we describe the molecular determinants for chemical activation of TRPV3 by the agonist carvacrol. Patch clamp recordings reveal that carvacrol activates TRPV3 in a concentration-dependent manner, with an EC50 of 0.2 mM, by increasing the probability of single-channel open conformation. Molecular docking of carvacrol into cryo-EM structure of TRPV3 combined with site-directed mutagenesis further identified a unique binding pocket formed by the channel S2-S3 linker important for mediating this interaction. Within the binding pocket consisting of four residues (Ile505, Leu508, Arg509, and Asp512), we report that Leu508 is the most critical residue for the activation of TRPV3 by carvacrol, but not 2-APB, a widely used nonspecific agonist and TRP channel modulator. Our findings demonstrate a direct binding of carvacrol to TRPV3 by targeting the channel S2-S3 linker that serves as a critical domain for chemical-mediated activation of TRPV3. We also propose that carvacrol can function as a molecular tool in the design of novel specific TRPV3 modulators for the further understanding of TRPV3 channel pharmacology. Transient receptor potential vanilloid 3 (TRPV3), robustly expressed in the skin, is a nonselective calcium-permeable cation channel activated by warm temperature, voltage, and certain chemicals. Natural monoterpenoid carvacrol from plant oregano is a known skin sensitizer or allergen that specifically activates TRPV3 channel. However, how carvacrol activates TRPV3 mechanistically remains to be understood. Here, we describe the molecular determinants for chemical activation of TRPV3 by the agonist carvacrol. Patch clamp recordings reveal that carvacrol activates TRPV3 in a concentration-dependent manner, with an EC50 of 0.2 mM, by increasing the probability of single-channel open conformation. Molecular docking of carvacrol into cryo-EM structure of TRPV3 combined with site-directed mutagenesis further identified a unique binding pocket formed by the channel S2-S3 linker important for mediating this interaction. Within the binding pocket consisting of four residues (Ile505, Leu508, Arg509, and Asp512), we report that Leu508 is the most critical residue for the activation of TRPV3 by carvacrol, but not 2-APB, a widely used nonspecific agonist and TRP channel modulator. Our findings demonstrate a direct binding of carvacrol to TRPV3 by targeting the channel S2-S3 linker that serves as a critical domain for chemical-mediated activation of TRPV3. We also propose that carvacrol can function as a molecular tool in the design of novel specific TRPV3 modulators for the further understanding of TRPV3 channel pharmacology. As a member of transient receptor potential vanilloid (TRPV) cation channel subfamily, the multimodal and nonselective calcium-permeable TRPV3 is robustly expressed in the skin and is activated by innocuous temperature at 31 to 33 °C, membrane potential, and chemical ligands (1Peier A.M. Reeve A.J. Andersson D.A. Moqrich A. Earley T.J. Hergarden A.C. Story G.M. Colley S. Hogenesch J.B. McIntyre P. Bevan S. Patapoutian A. A heat-sensitive TRP channel expressed in keratinocytes.Science. 2002; 296: 2046-2049Google Scholar, 2Smith G.D. Gunthorpe M.J. Kelsell R.E. Hayes P.D. Reilly P. Facer P. Wright J.E. Jerman J.C. Walhin J.P. Ooi L. 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Influence of TRPV3 mutation on hair growth cycle in mice.Biochem. Biophys. Res. Commun. 2007; 363: 479-483Google Scholar, 6Yoshioka T. Imura K. Asakawa M. Suzuki M. Oshima I. Hirasawa T. Sakata T. Horikawa T. Arimura A. Impact of the Gly573Ser substitution in TRPV3 on the development of allergic and pruritic dermatitis in mice.J. Invest. Dermatol. 2009; 129: 714-722Google Scholar, 7Moqrich A. Hwang S.W. Earley T.J. Petrus M.J. Murray A.N. Spencer K.S. Andahazy M. Story G.M. Patapoutian A. Impaired thermosensation in mice lacking TRPV3, a heat and camphor sensor in the skin.Science. 2005; 307: 1468-1472Google Scholar). Genetic gain-of-function mutations of TRPV3 cause a rare congenital disorder of Olmsted syndrome characterized by palmoplantar, periorificial keratoderma, alopecia, and severe itch (8Lai-Cheong J.E. Sethuraman G. Ramam M. Stone K. Simpson M.A. McGrath J.A. Recurrent heterozygous missense mutation, p.Gly573Ser, in the TRPV3 gene in an Indian boy with sporadic Olmsted syndrome.Br. J. Dermatol. 2012; 167: 440-442Google Scholar, 9Lin Z. Chen Q. Lee M. Cao X. Zhang J. Ma D. Chen L. Hu X. Wang H. Wang X. Zhang P. Liu X. Guan L. Tang Y. Yang H. et al.Exome sequencing reveals mutations in TRPV3 as a cause of Olmsted syndrome.Am. J. Hum. Genet. 2012; 90: 558-564Google Scholar, 10Danso-Abeam D. Zhang J. Dooley J. Staats K.A. Van Eyck L. Van Brussel T. Zaman S. Hauben E. Van de Velde M. Morren M.A. Renard M. Van Geet C. Schaballie H. Lambrechts D. Tao J. et al.Olmsted syndrome: Exploration of the immunological phenotype.Orphanet J. Rare Dis. 2013; 8: 79Google Scholar, 11Duchatelet S. Pruvost S. de Veer S. Fraitag S. Nitschke P. Bole-Feysot C. Bodemer C. Hovnanian A. A new TRPV3 missense mutation in a patient with Olmsted syndrome and erythromelalgia.JAMA Dermatol. 2014; 150: 303-306Google Scholar, 12Eytan O. Fuchs-Telem D. Mevorach B. Indelman M. Bergman R. Sarig O. Goldberg I. Adir N. Sprecher E. Olmsted syndrome caused by a homozygous recessive mutation in TRPV3.J. Invest. Dermatol. 2014; 134: 1752-1754Google Scholar, 13He Y. Zeng K. Zhang X. Chen Q. Wu J. Li H. Zhou Y. Glusman G. Roach J. Etheridge A. Qing S. Tian Q. Lee I. Tian X. Wang X. et al.A gain-of-function mutation in TRPV3 causes focal palmoplantar keratoderma in a Chinese family.J. Invest. Dermatol. 2014; 135: 907-909Google Scholar, 14Kariminejad A. Barzegar M. Abdollahimajd F. Pramanik R. McGrath J.A. Olmsted syndrome in an Iranian boy with a new de novo mutation in TRPV3.Clin. Exp. Dermatol. 2014; 39: 492-495Google Scholar), suggesting that TRPV3 may serve as a therapeutic target for pruritus and skin-related diseases. TRPV3 forms a tetrameric channel complex with each subunit consisting of six transmembrane-spanning segments (S1-S6), a pore-forming loop between S5 and S6, and cytosolic amino- (N-) and carboxy- (C-) termini (15Shioya T. Sato K. Sano M. Watanabe H. Transient receptor potential (TRP) channel and cough.Nihon Yakurigaku Zasshi. 2008; 131: 417-422Google Scholar). The ion permeation pathway of TRPV3 is lined by the S6 helices and the extended portions of the pore loops, including selectivity filter, central cavity, and gates (16Zubcevic L. Herzik M.A. Wu M. Borschel W.F. Hirschi M. Song A.S. Lander G.C. Lee S.-Y. Conformational ensemble of the human TRPV3 ion channel.Nat. Commun. 2018; 9: 4773Google Scholar). The S6 methionine residues (M677) form the intracellular gate with their side chains organized as a hydrophobic seal facing toward the center of ion permeation pathway (17Singh A.K. McGoldrick L.L. Sobolevsky A.I. Structure and gating mechanism of the transient receptor potential channel TRPV3.Nat. Struct. Mol. Biol. 2018; 25: 805-813Google Scholar). Unlike the gating rearrangements in TRPV1 (18Liao M. Cao E. Julius D. Cheng Y. Structure of the TRPV1 ion channel determined by electron cryo-microscopy.Nature. 2013; 504: 107-112Google Scholar, 19Gao Y. Cao E. Julius D. Cheng Y. TRPV1 structures in nanodiscs reveal mechanisms of ligand and lipid action.Nature. 2016; 534: 347-351Google Scholar) and TRPV6 (20McGoldrick L.L. Singh A.K. Saotome K. Yelshanskaya M.V. Twomey E.C. Grassucci R.A. Sobolevsky A.I. Opening of the human epithelial calcium channel TRPV6.Nature. 2018; 553: 233-237Google Scholar), upon heat or agonist binding, both the outer pore and the intracellular gate alone with conformational changes propagate to the TRP helix and result in overall conformational changes in TRPV3, especially the S1-S4 domains and the intracellular skirt rotating around the pore domain and expelling the lipids (17Singh A.K. McGoldrick L.L. Sobolevsky A.I. Structure and gating mechanism of the transient receptor potential channel TRPV3.Nat. Struct. Mol. Biol. 2018; 25: 805-813Google Scholar). The S1-S4 domain structural rearrangements underline the distinct gating mechanism of TRPV3 function (17Singh A.K. McGoldrick L.L. Sobolevsky A.I. Structure and gating mechanism of the transient receptor potential channel TRPV3.Nat. Struct. Mol. Biol. 2018; 25: 805-813Google Scholar). TRPV3 is activated by a variety of chemicals, including natural compounds such as camphor, carvacrol, thymol, synthesized 2-aminoethyl diphenylborinate (2-APB), and intracellular protons (21Cao X. Yang F. Zheng J. Wang K. Intracellular proton-mediated activation of TRPV3 channels accounts for the exfoliation effect of α-hydroxyl acids on keratinocytes.J. Biol. Chem. 2012; 287: 25905-25916Google Scholar, 22Chung M.K. Lee H. Mizuno A. Suzuki M. Caterina M.J. 2-Aminoethoxydiphenyl borate activates and sensitizes the heat-gated ion channel TRPV3.J. Neurosci. 2004; 24: 5177-5182Google Scholar, 23Xu H. Delling M. Jun J.C. Clapham D.E. Oregano, thyme and clove-derived flavors and skin sensitizers activate specific TRP channels.Nat. Neurosci. 2006; 9: 628-635Google Scholar). As a widely used tool molecule, 2-APB not only activates TRPV3 or other members of TRPs such as TRPV1, TRPV2, TRPA1, and TRP melastatin 6 (TRPM6) (24Hu H.Z. Gu Q. Wang C. Colton C.K. Tang J. Kinoshita-Kawada M. Lee L.Y. Wood J.D. Zhu M.X. 2-Aminoethoxydiphenyl borate is a common activator of TRPV1, TRPV2, and TRPV3.J. Biol. Chem. 2004; 279: 35741-35748Google Scholar, 25Hinman A. Chuang H.H. Bautista D.M. Julius D. TRP channel activation by reversible covalent modification.Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 19564-19568Google Scholar, 26Clapham D.E. SnapShot: Mammalian TRP channels.Cell. 2007; 129: 220Google Scholar) but also inhibits TRPM2, TRPM8, TRP canonical 4 (TRPC4), TRPC5, and TRPC6 channels (27Lievremont J.P. Bird G.S. Putney Jr., J.W. Mechanism of inhibition of TRPC cation channels by 2-aminoethoxydiphenylborane.Mol. Pharmacol. 2005; 68: 758-762Google Scholar, 28Togashi K. Inada H. Tominaga M. Inhibition of the transient receptor potential cation channel TRPM2 by 2-aminoethoxydiphenyl borate (2-APB).Br. J. Pharmacol. 2008; 153: 1324-1330Google Scholar, 29Kuhn F.J.P. Mathis W. Cornelia K. Hoffmann D.C. Luckhoff A. Modulation of activation and inactivation by Ca(2+) and 2-APB in the pore of an archetypal TRPM channel from Nematostella vectensis.Sci. Rep. 2017; 7: 7245Google Scholar). Chemical activation of TRPV3 is featured of different gating mechanisms. Electrophysiological characterizations confirm that two residues H426 in pre-S1 and R696 in the TRP helix are specifically required for TRPV3 sensitivity to 2-APB, but not camphor or voltage (23Xu H. Delling M. Jun J.C. Clapham D.E. Oregano, thyme and clove-derived flavors and skin sensitizers activate specific TRP channels.Nat. Neurosci. 2006; 9: 628-635Google Scholar, 30Vogt-Eisele A.K. Weber K. Sherkheli M.A. Vielhaber G. Panten J. Gisselmann G. Hatt H. Monoterpenoid agonists of TRPV3.Br. J. Pharmacol. 2007; 151: 530-540Google Scholar, 31Hu H. Grandl J. Bandell M. Petrus M. Patapoutian A. Two amino acid residues determine 2-APB sensitivity of the ion channels TRPV3 and TRPV4.Proc. Natl. Acad. Sci. U. S. A. 2009; 106: 1626-1631Google Scholar). Camphor also activates TRPV3 with an EC50 about 6 mM, involving the S4-S5 linker for its binding (30Vogt-Eisele A.K. Weber K. Sherkheli M.A. Vielhaber G. Panten J. Gisselmann G. Hatt H. Monoterpenoid agonists of TRPV3.Br. J. Pharmacol. 2007; 151: 530-540Google Scholar, 32Nguyen T.H.D. Itoh S.G. Okumura H. Tominaga M. Structural basis for promiscuous action of monoterpenes on TRP channels.Commun. Biol. 2021; 4: 293Google Scholar). Natural monoterpenoid carvacrol (5-Isopropyl-2-methylphenol) as a skin sensitizer is a major component of plant oregano and has been shown to activate TRPV3 with an EC50 of (23Xu H. Delling M. Jun J.C. Clapham D.E. Oregano, thyme and clove-derived flavors and skin sensitizers activate specific TRP channels.Nat. Neurosci. 2006; 9: 628-635Google Scholar, 30Vogt-Eisele A.K. Weber K. Sherkheli M.A. Vielhaber G. Panten J. Gisselmann G. Hatt H. Monoterpenoid agonists of TRPV3.Br. J. Pharmacol. 2007; 151: 530-540Google Scholar). also activates and causes the channel (23Xu H. Delling M. Jun J.C. Clapham D.E. Oregano, thyme and clove-derived flavors and skin sensitizers activate specific TRP channels.Nat. Neurosci. 2006; 9: 628-635Google Scholar). Our that activation of TRPV3 by carvacrol pruritus in but not in mice Wang Wang K. A for the activation of TRPV3 channel in itch by the natural skin sensitizer Pharmacol. 2018; 39: Scholar, Y. Wang G. X. Wang K. Inhibition of the warm transient receptor potential vanilloid TRPV3 channel Pharmacol. Scholar). TRPV3 natural and of by carvacrol H. X. H. Ma Q. Zhou Q. Wang W. Wang K. inhibition of the temperature-sensitive and transient receptor potential vanilloid TRPV3 channel by natural pruritus and of keratinocytes.J. Pharmacol. Exp. Scholar). However, how TRPV3 is activated by agonist carvacrol remains the molecular mechanism for specific activation of TRPV3 by carvacrol may to of specific modulators for the of TRPV3 pharmacology. this we the molecular the chemical activation of TRPV3 by specific agonist carvacrol combined of molecular and site-directed Our findings reveal that carvacrol to a pocket formed by the S2-S3 and to the activation of TRPV3 demonstrate the direct action of carvacrol on TRPV3, we TRPV3 activation in to different of carvacrol. As shown in carvacrol activated the of TRPV3 channels in a concentration-dependent with an EC50 of single-channel recordings at from to that carvacrol at also caused a activation of TRPV3 with a of to the used agonist 2-APB that also activated TRPV3 channels with a at further the effect of carvacrol on TRPV3 we the single-channel recordings in at or 2-APB as TRPV3 single-channel open TRPV3 the channel A and that carvacrol to an of to and an open to as with the at the to and open to 2-APB at to an of to and an open to as with the at the to and open to demonstrate that carvacrol on TRPV3 at and single-channel the between carvacrol and TRPV3, we the molecular docking and the of binding from the of binding A and and The pocket and formed by four helices S1-S4 are the membrane and the and The pocket 3 is at the pre-S1 helix and the TRP helix domain to 3 are involved in activation of TRPV3 (17Singh A.K. McGoldrick L.L. Sobolevsky A.I. Structure and gating mechanism of the transient receptor potential channel TRPV3.Nat. Struct. Mol. Biol. 2018; 25: 805-813Google Scholar). The pocket 4 is formed between helix and helix and the pocket is formed by the S2-S3 linker the binding from docking reveals distinct between the pocket residues and carvacrol that the most to the ligand binding this we site-directed mutagenesis combined with the residues the with carvacrol in an of TRPV3 activation the pocket but not mutations in the to 4 that the channel activation by carvacrol, as to TRPV3 demonstrate that the pocket formed by the S2-S3 linker is an important domain for TRPV3 The chemical structure of carvacrol is featured of an with two a a and the further confirm the residue in the pocket critical for binding to the of carvacrol by the we the residue into different amino acids and the channel sensitivity to carvacrol. Molecular docking shows an for carvacrol with its and on of the pocket in TRPV3 in a with the of residue or single-channel recordings of TRPV3 further that the channel to different of carvacrol or 2-APB at and mutation of to in a of with the of carvacrol and a of the between carvacrol and or single-channel recordings of TRPV3 further the of the channel activation by carvacrol, but not 2-APB that to activate the channel and to the of the pocket also TRPV3 activation by carvacrol, but not 2-APB and to a also to the channel activation of TRPV3 by carvacrol with its and the on the two on the pocket as the ligand to form the with by that the of the between carvacrol and is critical for chemical activation of TRPV3. on residue with its its the two residues and with its binding to residue a between the and residue is also involved in the binding A and The with residue and a with to to a of the channel sensitivity to carvacrol that only caused a activation of TRPV3 at of 3 mM, as with TRPV3 or an activation of TRPV3 by 2-APB to in a of and sensitivity to 2-APB The of carvacrol with the residue and the mutation also the channel sensitivity to carvacrol at both and single-channel the of carvacrol to the residue and the mutation also the sensitivity to carvacrol with the activation of TRPV3 by carvacrol with an EC50 at mM, and in the of the channel activation to for with EC50 of mM, mM, and mM, and demonstrate that the with residues and the domain of S2-S3 are critical for activation of TRPV3 channel. The of this to the molecular mechanism of action for natural carvacrol that specifically activates TRPV3 in with a and widely used agonist a of molecular docking and site-directed we that the agonist carvacrol activates TRPV3 a direct action on the target at the channel We also a binding pocket formed by four amino acids and the S2-S3 linker that the chemical activation of TRPV3 by carvacrol. TRPV3 different in to chemical or heat A.K. McGoldrick L.L. L. M. E. Sobolevsky A.I. Structural basis of temperature by the TRP channel TRPV3.Nat. Struct. Mol. Biol. Scholar, A. N. E. Sobolevsky A.I. Structural mechanism of of a temperature-sensitive TRP channel.Nat. Struct. Mol. Biol. 2021; Scholar, Q. Wang J. X. Hu J. C. Y. Xie C. Wang P. Cao P. Cao Z. Y. Li D. J. inhibition of TRPV3 channel by 2021; Scholar). at activates TRPV3 with a at A.K. McGoldrick L.L. L. M. E. Sobolevsky A.I. Structural basis of temperature by the TRP channel TRPV3.Nat. Struct. Mol. Biol. Scholar, A. N. E. Sobolevsky A.I. Structural mechanism of of a temperature-sensitive TRP channel.Nat. Struct. Mol. Biol. 2021; Scholar). Chemical activation of TRPV3 by 2-APB in the or of TRPV3 also reveals a different of and Q. Wang J. X. Hu J. C. Y. Xie C. Wang P. Cao P. Cao Z. Y. Li D. J. inhibition of TRPV3 channel by 2021; Scholar). this single-channel recordings of TRPV3 activation by carvacrol and 2-APB at from to a of and The cryo-EM structures of TRPV3 reveal that agonist 2-APB to a pocket formed by the of S1-S4 domains (17Singh A.K. McGoldrick L.L. Sobolevsky A.I. Structure and gating mechanism of the transient receptor potential channel TRPV3.Nat. Struct. Mol. Biol. 2018; 25: 805-813Google Scholar), and the between S1-S4 and the pore domain is the of S5 and S6 from the pore with an overall of TRPV3 (17Singh A.K. McGoldrick L.L. Sobolevsky A.I. Structure and gating mechanism of the transient receptor potential channel TRPV3.Nat. Struct. Mol. Biol. 2018; 25: 805-813Google Scholar). this we potential binding from and identified the residues and TRPV3 S2-S3 linker domain that channel with a residue in the S2-S3 a conformational channel The S2-S3 linker has been shown to an important in multimodal gating of TRPV1 that about with TRPV3 D.E. TRP channels as Scholar, C. The TRP of cation 2005; Scholar, A. Patapoutian A. ion channels and temperature Neurosci. 2006; Scholar). activation of TRPV1 channel is involved in linker domains such as the membrane domain the S2-S3 and the S4-S5 to the heat or channel activation S. K.J. with TRPV1 channels in a lipid Molecular J. Scholar, H. F. Zheng W. the heat activation mechanism of the TRPV1 channel gating by molecular 2016; Scholar, H. Zheng W. the heat activation mechanism of TRPV1 channel by molecular J. 2018; Scholar, W. H. activation mechanism of from molecular Scholar). A shows that of TRPV3 are by a conformational involving structural changes of the structure and of the S2-S3 linker and the and termini for the channel and temperature A. N. E. Sobolevsky A.I. Structural mechanism of of a temperature-sensitive TRP channel.Nat. Struct. Mol. Biol. 2021; Scholar). chemical activation of TRPV3, we that of the important residues the S2-S3 a critical in chemical activation of TRPV3 by carvacrol with its both and as two for binding and the channel open conformation. the of carvacrol cause the of S2-S3 domain by in the and in the in with the between the and the residue a or can to conformational changes in a in the pore The of carvacrol as is for the of the critical for TRPV3 with carvacrol, the monoterpenoid agonists the or the can only or TRPV3 (30Vogt-Eisele A.K. Weber K. Sherkheli M.A. Vielhaber G. Panten J. Gisselmann G. Hatt H. Monoterpenoid agonists of TRPV3.Br. J. Pharmacol. 2007; 151: 530-540Google Scholar). findings a direct action of natural carvacrol on TRPV3 channels a binding pocket formed by the S2-S3 The of the agonist pocket in TRPV3 S2-S3 linker not only the gating of the channel but also serves as a target for further of novel TRPV3 modulators with and mutations on human TRPV3 by used to mutations are in mutations in with and in an at in an with and Transient by human TRPV3 in and into at temperature for The to and 2-APB from Natural from and camphor from and at of carvacrol 2-APB and camphor in the and to a of 3 to 4 a The the The the Transient receptor potential vanilloid 3 channel in at in to a voltage from to for and the at clamp recordings at temperature recordings a by Patch and to a of to 6 a The and 3 used in both and potential at and at and at clamp recordings at temperature The between carvacrol and TRPV3 cryo-EM structure molecular docking The structure of carvacrol from the the G.M. R. A.J. docking a and an binding Chem. Scholar). The used in the docking for ligand conformational to the docking the the binding to in used for amino acid mutation, and is used to the with and and used to the EC50 determined by to are expressed as the and for between two A of is to be are the A. N. E. Sobolevsky A.I. Structural mechanism of of a temperature-sensitive TRP channel.Nat. Struct. Mol. Biol. 2021; Scholar). The of in this by Natural of and and the of and of C. F. X. and K. W. C. N. C. X. F. and X. T. C. X. F. X. and K. W. C. N. X. T. and K. W. K. W. C. X. F. X. and K. W. and K. W. K. W. K. W. C. N. with with with and

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