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; : Activation of the TRPV4 ion channel is enhanced by phosphorylation
Hueng-Chuen Fan1,2, Xuming Zhang1, PeterAMcNaughton1*
From 1Department of Pharmacology, University of Cambridge, Tennis Court Rd, Cambridge CB2 1PD, UK; 2Department of Pediatrics, Tri-Service General Hospital and National Defense Medical Center, Taipei, Taiwan, ROC
Running head: Activation of TRPV4 is enhanced by phosphorylation
*Address correspondence to: PeterAMcNaughton, Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1PD, UK. Tel.: +44 1223 334012; Fax: +44 1223 334100; E-mail: HYPERLINK "mailto:pam42@cam.ac.uk" \t "_top" pam42@cam.ac.uk
The TRPV4 (transient receptor potential vanilloid 4) ion channel, a member of the vanilloid subfamily of the TRP channels, is activated by membrane stretch, by non-noxious warm temperatures, and by a range of chemical activators. In the present study we examined the role of phosphorylation in modulating activation of TRPV4. We expressed TRPV4 in HEK293 cells and activated the channel by cell swelling in a hypotonic solution. TRPV4 channel activation and serine phosphorylation were enhanced by exposure to the PKC activator phorbol 12-myristate 13-acetate (PMA) or by application of bradykinin, which activates PKC via a G-protein coupled mechanism. The enhancement was inhibited by the PKC inhibitors staurosporine, BIM, and rottlerin, or by mutation of the serine/threonine residues S162, T175 and S189. The adenylate cyclase activator forskolin (FSK) also enhanced activation of TRPV4, and the enhancement was antagonized by the selective PKA inhibitor H89 or by mutation of serine residue S824. Sensitization of TRPV4 by both PKC and PKA depended on the scaffolding protein AKAP79, because channel activation and phosphorylation were enhanced by co-transfection of AKAP79 and were antagonized by removal of AKAP79 using siRNA. We conclude that the S/T kinases PKC and PKA enhance activation of the TRPV4 ion channel by phosphorylation at specific sites, and that phosphorylation depends on assembly of PKC and PKA by AKAP79 into a signaling complex with TRPV4.
TRPV4 was cloned from kidney, hypothalamus and auditory epithelium and was given a number of names: Osm-9-like TRP channel 4 (OTRPC4) ADDIN REFMGR.CITE Strotmann2000318OTRPC4, a nonselective cation channel that confers sensitivity to extracellular osmolarityJournal318OTRPC4, a nonselective cation channel that confers sensitivity to extracellular osmolarityStrotmann,R.Harteneck,C.Nunnenmacher,K.Schultz,G.Plant,T.D.2000/10Amino Acid SequenceAnimalsCalciumCation Transport ProteinsCationschemistryComparative StudyElectric ConductivityElectrophysiologygeneticsHeartIon Channelsisolation & purificationKidneyLivermetabolismMiceMolecular Sequence DataMyocardiumOsmotic PressurePatch-Clamp TechniquesProteinsSequence Homology,Amino AcidSignal TransductionTissue DistributionTRPV Cation ChannelsNot in File695702Nat.Cell Biol.210Institut fur Pharmakologie, Universitatsklinikum Benjamin Franklin, Freie Universitat Berlin, Thielallee 67-73, 14195 Berlin, GermanyPM:11025659Nat.Cell Biol.1(1), VR-OAC ADDIN REFMGR.CITE Liedtke2000319Vanilloid receptor-related osmotically activated channel (VR-OAC), a candidate vertebrate osmoreceptorJournal319Vanilloid receptor-related osmotically activated channel (VR-OAC), a candidate vertebrate osmoreceptorLiedtke,W.Choe,Y.Marti-Renom,M.A.Bell,A.M.Denis,C.S.Sali,A.Hudspeth,A.J.Friedman,J.M.Heller,S.2000/10/27Amino Acid SequenceanalysisAnimalsAnkyrin RepeatBrainCalcium SignalingCationsCentral Nervous SystemchemistryChickensCho CellsChromosomes,Human,Pair 12Chromosomes,Human,Pair 17Cloning,MolecularCricetinaecytologyElectrophysiologyGene Expression ProfilingGenesgeneticsHairHair Cells,Auditory,InnerHumanHumansHypotonic SolutionsIn Situ HybridizationIon Channel GatingIon ChannelsMerkel CellsmetabolismMiceMolecular Sequence DataNervous SystemNeuronsNeurons,AfferentOsmolar ConcentrationOsmotic PressurePhylogenyphysiologyProteinsRadiation Hybrid MappingRatsReceptors,DrugResearchRnaRNA,MessengerSequence AlignmentNot in File525535Cell1033Laboratory of Molecular Genetics, The Rockefeller University, New York, New York 10021, USAPM:11081638Cell1(2), TRP12 ADDIN REFMGR.CITE Wissenbach2000320Trp12, a novel Trp related protein from kidneyJournal320Trp12, a novel Trp related protein from kidneyWissenbach,U.Bodding,M.Freichel,M.Flockerzi,V.2000/11/24Amino Acid SequenceanalysisAnimalsCalciumCalcium ChannelsCell LinechemistryCho CellsChromosomes,Human,Pair 12Cloning,MolecularCodonCricetinaeCytosolDnaExonsGene ExpressiongeneticsHumanHumansHypotonic SolutionsIntronsIon ChannelsKidneymetabolismMiceMolecular Sequence DatapharmacologyphysiologyPolymerase Chain ReactionResearchRnaRNA SplicingRNA,MessengerSequence HomologyTransfectionTRPC Cation ChannelsNot in File127134FEBS Lett.4852-3Institut fur Pharmakologie und Toxikologie der Universitat des Saarlandes, D-66421 Homburg, Germany. ulrich.wissenbach@med-rz.uni-saarland.dePM:11094154FEBS Lett.1(3) and vanilloid receptor-like channel 2 (VRL-2) ADDIN REFMGR.CITE Delany2001321Identification and characterization of a novel human vanilloid receptor-like protein, VRL-2Journal321Identification and characterization of a novel human vanilloid receptor-like protein, VRL-2Delany,N.S.Hurle,M.Facer,P.Alnadaf,T.Plumpton,C.Kinghorn,I.See,C.G.Costigan,M.Anand,P.Woolf,C.J.Crowther,D.Sanseau,P.Tate,S.N.2001/1/19Amino Acid SequenceanalysisAnimalsAntibodiesbloodBlotting,NorthernCapsaicinCation Transport ProteinsCell LinechemistryChromosome MappingChromosomes,Human,Pair 12Cloning,MolecularComparative StudyDnaDNA,ComplementaryFemaleGangliaGenesgeneticsHumanHumansImmunohistochemistryIn Situ Hybridization,FluorescenceIon Channelsisolation & purificationKidneyMalemetabolismMolecular Sequence DataNerve FibersNervous SystemProteinsRadiation Hybrid MappingRatsReceptors,DrugResearchRnaSequence AlignmentSequence Analysis,DNASequence Homology,Amino AcidSkinTissue DistributionTRPV Cation ChannelsNot in File165174Physiol Genomics43Genome Informatics and Analysis, Virology and Vaccine Systems, Ion Channel Section, Molecular Recognition, Molecular Genetics, Glaxo Wellcome Research and Development, Medicines Research Centre, Stevenage, Hertfordshire SG1 2NY, United KingdomPM:11160995Physiol Genomics1(4). The gene for human TRPV4 is located on chromosome 12q23-q24.1 and has 15 exons, which code for a full-length protein with 871 amino acids. TRPV4 is a member of the TRPV subfamily of TRP channels, and like other members of this subfamily it is a polymodal receptor activated by a wide variety of stimuli. TRPV4 is strongly expressed in kidney and is activated by hypotonicity, which has led to the suggestion that TRPV4 is an osmosensor important in regulating body fluid levels ADDIN REFMGR.CITE Liedtke2000319Vanilloid receptor-related osmotically activated channel (VR-OAC), a candidate vertebrate osmoreceptorJournal319Vanilloid receptor-related osmotically activated channel (VR-OAC), a candidate vertebrate osmoreceptorLiedtke,W.Choe,Y.Marti-Renom,M.A.Bell,A.M.Denis,C.S.Sali,A.Hudspeth,A.J.Friedman,J.M.Heller,S.2000/10/27Amino Acid SequenceanalysisAnimalsAnkyrin RepeatBrainCalcium SignalingCationsCentral Nervous SystemchemistryChickensCho CellsChromosomes,Human,Pair 12Chromosomes,Human,Pair 17Cloning,MolecularCricetinaecytologyElectrophysiologyGene Expression ProfilingGenesgeneticsHairHair Cells,Auditory,InnerHumanHumansHypotonic SolutionsIn Situ HybridizationIon Channel GatingIon ChannelsMerkel CellsmetabolismMiceMolecular Sequence DataNervous SystemNeuronsNeurons,AfferentOsmolar ConcentrationOsmotic PressurePhylogenyphysiologyProteinsRadiation Hybrid MappingRatsReceptors,DrugResearchRnaRNA,MessengerSequence AlignmentNot in File525535Cell1033Laboratory of Molecular Genetics, The Rockefeller University, New York, New York 10021, USAPM:11081638Cell1Alessandri-Haber2003357Hypotonicity induces TRPV4-mediated nociception in ratJournal357Hypotonicity induces TRPV4-mediated nociception in ratAlessandri-Haber,N.Yeh,J.J.Boyd,A.E.Parada,C.A.Chen,X.Reichling,D.B.Levine,J.D.2003/7/31Afferent PathwaysAnalgesicsAnimalsantagonists & inhibitorsBase SequenceBehaviorCation Transport Proteinsdrug effectsExtracellular SpaceHamstersHypotonic SolutionsIon ChannelsMalemetabolismmethodsMolecular Sequence DataNerve EndingsNerve FibersNeuronsOsmolar ConcentrationPain MeasurementPatch-Clamp TechniquesphysiologyProteinsRatsRats,Sprague-DawleyResearch Support,U.S.Gov't,P.H.S.SkinSolutionsNot in File497511Neuron393Division of Neuroscience, University of California, San Francisco, San Francisco, California 94143, USAPM:12895423Neuron1Gao2003368Temperature-modulated diversity of TRPV4 channel gating: activation by physical stresses and phorbol ester derivatives through protein kinase C-dependent and -independent pathwaysJournal368Temperature-modulated diversity of TRPV4 channel gating: activation by physical stresses and phorbol ester derivatives through protein kinase C-dependent and -independent pathwaysGao,X.Wu,L.O'neil,R.G.2003/7/18analysisAnimalsCalciumCation Transport ProteinsCell LineCho CellsCloning,MolecularCyclic AMP-Dependent Protein Kinasesdrug effectsFura-2geneticsHamstersHumanHumansIon Channel GatingIon ChannelsKidneymetabolismMicePatch-Clamp TechniquespharmacologyPhorbol EstersProtein KinasesProteinsRecombinant ProteinsResearch Support,Non-U.S.Gov'tResearch Support,U.S.Gov't,P.H.S.Ruthenium RedSignal TransductionStaurosporineTemperatureTetradecanoylphorbol AcetateTransfectionNot in File2712927137J.Biol.Chem.27829Department of Integrative Biology and Pharmacology, The University of Texas Health Science Center at Houston, Houston, Texas 77030, USAPM:12738791J.Biol.Chem.1Nilius2001348Differential activation of the volume-sensitive cation channel TRP12 (OTRPC4) and volume-regulated anion currents in HEK-293 cellsJournal348Differential activation of the volume-sensitive cation channel TRP12 (OTRPC4) and volume-regulated anion currents in HEK-293 cellsNilius,B.Prenen,J.Wissenbach,U.Bodding,M.Droogmans,G.2001/11AnionsCation Transport ProteinsCationsCell LinecytologyDown-RegulationElectric ConductivityGuanosine 5'-O-(3-Thiotriphosphate)HumanHumansIon ChannelsIonsmetabolismPermeabilitypharmacologyphysiologyProteinsResearch Support,Non-U.S.Gov'tTransfectionNot in File227233Pflugers Arch.4432Department of Physiology, Campus Gasthuisberg, KU Leuven, Herestraat 49, 3000 Leuven, Belgium. bernd.nilius@med.kuleuven.ac.bePM:11713648Pflugers Arch.1Vriens2004362Cell swelling, heat, and chemical agonists use distinct pathways for the activation of the cation channel TRPV4Journal362Cell swelling, heat, and chemical agonists use distinct pathways for the activation of the cation channel TRPV4Vriens,J.Watanabe,H.Janssens,A.Droogmans,G.Voets,T.Nilius,B.2004/1/6agonistsAlanineAnimalsantagonists & inhibitorsArachidonic AcidBrainCation Transport ProteinsCationsCell LinechemistryCytochrome P-450 Enzyme SystemEndotheliumEnzyme InhibitorsgeneticsHeatHumanHumansIon ChannelsmetabolismMiceMutagenesis,Site-DirectedOsmotic PressureOxygenasespharmacologyPhorbol EstersPhospholipases APhosphorylationphysiologyProteinsRecombinant ProteinsResearch Support,Non-U.S.Gov'tSignal TransductionTyrosineNot in File396401Proc.Natl.Acad.Sci.U.S.A1011Department of Physiology, Campus Gasthuisberg, Katholieke Universiteit Leuven, B-3000 Leuven, BelgiumPM:14691263Proc.Natl.Acad.Sci.U.S.A1Birder20074073Activation of urothelial transient receptor potential vanilloid 4 by 4alpha-phorbol 12,13-didecanoate contributes to altered bladder reflexes in the ratJournal4073Activation of urothelial transient receptor potential vanilloid 4 by 4alpha-phorbol 12,13-didecanoate contributes to altered bladder reflexes in the ratBirder,L.Kullmann,F.A.Lee,H.Barrick,S.de Groat,W.Kanai,A.Caterina,M.2007/10AACTIVATIONagonistsAnimalsarticleAsbiosynthesisBladderCapsaicincellCellsCHANNELdrug effectsFemaleHeatImmunoblottingImmunohistochemistryIn Vitroion channelMalemediatormetabolismMuscle ContractionMuscle,SmoothOrpharmacologyPhorbol EstersphysiologyRatsRats,Sprague-DawleyReflexResearchReverse Transcriptase Polymerase Chain ReactionTRPV Cation ChannelsUrinary BladderUrinationUrotheliumNot in File227235J Pharmacol.Exp.Ther.3231Department of Medicine, University of Pittsburgh School of Medicine, A 1207 Scaife Hall, Pittsburgh, PA 15261, USA. lbirder@pitt.eduPM:17636010J Pharmacol.Exp.Ther.1(5-10). However, TRPV4 is also activated by innocuous heat with a threshold > 27(C ADDIN REFMGR.CITE Gao2003368Temperature-modulated diversity of TRPV4 channel gating: activation by physical stresses and phorbol ester derivatives through protein kinase C-dependent and -independent pathwaysJournal368Temperature-modulated diversity of TRPV4 channel gating: activation by physical stresses and phorbol ester derivatives through protein kinase C-dependent and -independent pathwaysGao,X.Wu,L.O'neil,R.G.2003/7/18analysisAnimalsCalciumCation Transport ProteinsCell LineCho CellsCloning,MolecularCyclic AMP-Dependent Protein Kinasesdrug effectsFura-2geneticsHamstersHumanHumansIon Channel GatingIon ChannelsKidneymetabolismMicePatch-Clamp TechniquespharmacologyPhorbol EstersProtein KinasesProteinsRecombinant ProteinsResearch Support,Non-U.S.Gov'tResearch Support,U.S.Gov't,P.H.S.Ruthenium RedSignal TransductionStaurosporineTemperatureTetradecanoylphorbol AcetateTransfectionNot in File2712927137J.Biol.Chem.27829Department of Integrative Biology and Pharmacology, The University of Texas Health Science Center at Houston, Houston, Texas 77030, USAPM:12738791J.Biol.Chem.1Guler2002326Heat-evoked activation of the ion channel, TRPV4Journal326Heat-evoked activation of the ion channel, TRPV4Guler,A.D.Lee,H.Iida,T.Shimizu,I.Tominaga,M.Caterina,M.2002/8/1AnimalsBody Temperature RegulationCalciumCation Transport Proteinschemistrycytologydrug effectsEnzyme InhibitorsFluorescent DyesgeneticsHeatHomeostasisHumanHumansHypothalamus,AnteriorImmunohistochemistryIon ChannelsKidneymetabolismNervous SystemOocytesOsmolar ConcentrationPatch-Clamp TechniquespharmacologyphysiologyPreoptic AreaProteinsRatsResearchRnaRNA,MessengerRuthenium RedSensationTemperatureTransfectionTRPV Cation ChannelsXenopusXenopus laevisNot in File64086414J.Neurosci.2215Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USAPM:12151520J.Neurosci.1Watanabe200269Heat-evoked activation of TRPV4 channels in a HEK293 cell expression system and in native mouse aorta endothelial cellsJournal69Heat-evoked activation of TRPV4 channels in a HEK293 cell expression system and in native mouse aorta endothelial cellsWatanabe,H.Vriens,J.Suh,S.H.Benham,C.D.Droogmans,G.Nilius,B.2002/12/6AnimalsAortaCalciumCation Transport ProteinsCell LinecytologyElectrophysiologyEndotheliumEndothelium,VascularGene DeletionHeatHumanHumansIon ChannelsKineticsLigandsMembrane PotentialsmetabolismMicephysiologyProtein Structure,TertiaryProteinsResearch Support,Non-U.S.Gov'tSignal TransductionTemperatureTime FactorsTransfectionNot in File4704447051J.Biol.Chem.27749Department of Physiology, Campus Gasthuisberg, KU Leuven, B-3000 Leuven, BelgiumPM:12354759J.Biol.Chem.1(11-13), by the phorbol ester 4(-phorbol 12,13-didecanoate (4(PDD) ADDIN REFMGR.CITE Watanabe2002344Activation of TRPV4 channels (hVRL-2/mTRP12) by phorbol derivativesJournal344Activation of TRPV4 channels (hVRL-2/mTRP12) by phorbol derivativesWatanabe,H.Davis,J.B.Smart,D.Jerman,J.C.Smith,G.D.Hayes,P.Vriens,J.Cairns,W.Wissenbach,U.Prenen,J.Flockerzi,V.Droogmans,G.Benham,C.D.Nilius,B.2002/4/19agonistsAnimalsCalciumCation Transport ProteinsCationsCell LineCells,CulturedchemistryDose-Response Relationship,DrugDyesElectrophysiologyEndothelium,VascularHumanHumansIon ChannelsKineticsmetabolismMicepharmacologyPhorbolsphysiologyProtein Kinase CProteinsReceptors,DrugRecombinant ProteinsResearch Support,Non-U.S.Gov'tRuthenium RedTransfectionTumor Cells,CulturedNot in File1356913577J.Biol.Chem.27716Department of Physiology, Campus Gasthuisberg, KU Leuven, B-3000 Leuven, BelgiumPM:11827975J.Biol.Chem.1Xu2003374Protein kinase C-mediated Ca2+ entry in HEK 293 cells transiently expressing human TRPV4Journal374Protein kinase C-mediated Ca2+ entry in HEK 293 cells transiently expressing human TRPV4Xu,F.Satoh,E.Iijima,T.2003/9antagonists & inhibitorsCalciumCation Transport ProteinsCell LinechemistryComparative StudyDose-Response Relationship,Drugdrug effectsEnzyme ActivationEnzyme InhibitorsFura-2Gene ExpressiongeneticsHumanHumansHypotonic SolutionsIndolesIon ChannelsMaleimidesmetabolismpharmacologyPhorbol 12,13-DibutyratePhorbol EstersProtein Kinase CProteinsResearch Support,Non-U.S.Gov'tSolutionsTetradecanoylphorbol AcetateNot in File413421Br.J.Pharmacol.1402Department of Pharmacology, Akita University School of Medicine, 1-1-1 Hondoh, Akita 010-8543, JapanPM:12970074Br.J.Pharmacol.1(14;15), by low pH ADDIN REFMGR.CITE Suzuki2003355Impaired pressure sensation in mice lacking TRPV4Journal355Impaired pressure sensation in mice lacking TRPV4Suzuki,M.Mizuno,A.Kodaira,K.Imai,M.2003/6/20AnimalsBlotting,NorthernCapsaicinCation Transport ProteinsCho CellsdeficiencyGangliaGanglia,SpinalgeneticsHamstersHeatIn VitroIon ChannelsKidneyMechanoreceptorsMembrane PotentialsMiceMice,KnockoutMolecular Sequence DataNeuronsOsmotic PressurepharmacologyphysiologyProteinsResearch Support,Non-U.S.Gov'tSensationTransfectionVertebratesNot in File2266422668J.Biol.Chem.27825Department of Pharmacology, Jichi Medical School, 3311-1 Yakushiji, Minamikawachi, Tochigi 329-0498, JapanPM:12692122J.Biol.Chem.1(16), by endocannabinoids and arachidonic acid metabolites ADDIN REFMGR.CITE Vriens2005385Modulation of the Ca2 permeable cation channel TRPV4 by cytochrome P450 epoxygenases in vascular endotheliumJournal385Modulation of the Ca2 permeable cation channel TRPV4 by cytochrome P450 epoxygenases in vascular endotheliumVriens,J.Owsianik,G.Fisslthaler,B.Suzuki,M.Janssens,A.Voets,T.Morisseau,C.Hammock,B.D.Fleming,I.Busse,R.Nilius,B.2005/10/288,11,14-Eicosatrienoic AcidAcidsagonistsanalogs & derivativesAnimalsantagonists & inhibitorsArachidonic AcidCalciumCells,CulturedCytochrome P-450 Enzyme SystemEndothelial CellsEndotheliumEpoxide HydrolasesHeatHydrolysisIndomethacinmetabolismMiceNifedipinepharmacologyphysiologyResearchTemperatureTransient Receptor Potential ChannelsTRPV Cation ChannelsNot in File908915Circ.Res.979Department of Physiology, Campus Gasthuisberg, KU Leuven, BelgiumPM:16179585Circ.Res.1Watanabe2003346Anandamide and arachidonic acid use epoxyeicosatrienoic acids to activate TRPV4 channelsJournal346Anandamide and arachidonic acid use epoxyeicosatrienoic acids to activate TRPV4 channelsWatanabe,H.Vriens,J.Prenen,J.Droogmans,G.Voets,T.Nilius,B.2003/7/248,11,14-Eicosatrienoic AcidAcidsanalogs & derivativesAnimalsAortaArachidonic AcidArachidonic AcidsCalciumCation Transport ProteinsCell Linedrug effectsElectrophysiologyEndocannabinoidsEndothelium,VascularHeatHumanHumansIon Channel GatingIon ChannelsmetabolismMicepharmacologyphysiologyProteinsResearch Support,Non-U.S.Gov'tNot in File434438Nature4246947Department of Physiology, Campus Gasthuisberg, KU Leuven, B-3000 Leuven, BelgiumPM:12879072Nature1(17;18), by the active compound, bisandrographolide A, of Andrographis paniculata, a Chinese herbal plant ADDIN REFMGR.CITE Smith2006674Bisandrographolide from Andrographis paniculata activates TRPV4 channelsJournal674Bisandrographolide from Andrographis paniculata activates TRPV4 channelsSmith,P.L.Maloney,K.N.Pothen,R.G.Clardy,J.Clapham,D.E.2006/10/6AndrographisCapsaicinCell LineCellsDiterpenesHumanHumansKeratinocytesMedicine,Chinese TraditionalmetabolismResearchTRPV Cation ChannelsNot in File2989729904J.Biol.Chem.28140Department of Cardiology, Howard Hughes Medical Institute, Children's Hospital, Boston, Massachusetts 02115, USAPM:16899456J.Biol.Chem.1(19) and by nitric oxide ADDIN REFMGR.CITE Yoshida2006675Nitric oxide activates TRP channels by cysteine S-nitrosylationJournal675Nitric oxide activates TRP channels by cysteine S-nitrosylationYoshida,T.Inoue,R.Morii,T.Takahashi,N.Yamamoto,S.Hara,Y.Tominaga,M.Shimizu,S.Sato,Y.Mori,Y.2006/11AnimalsCalciumCalcium ChannelsCellsCells,CulturedchemistryChickensCysteineEndothelial CellsHumansmetabolismModels,MolecularMolecular StructureNitric OxideNitrogen CompoundsphysiologyProtein BindingProteinsResearchSensitivity and SpecificitySignal TransductionSulfhydryl CompoundsTime FactorsTransient Receptor Potential ChannelsNot in File596607Nat.Chem.Biol.211Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyoto 615-8510, JapanPM:16998480Nat.Chem.Biol.1(20). TRPV4 is expressed in a broad range of tissues, including lung, spleen, kidney, testis, fat, brain, cochlea, skin, smooth muscle, liver, vascular endothelium ADDIN REFMGR.CITE Liedtke2000319Vanilloid receptor-related osmotically activated channel (VR-OAC), a candidate vertebrate osmoreceptorJournal319Vanilloid receptor-related osmotically activated channel (VR-OAC), a candidate vertebrate osmoreceptorLiedtke,W.Choe,Y.Marti-Renom,M.A.Bell,A.M.Denis,C.S.Sali,A.Hudspeth,A.J.Friedman,J.M.Heller,S.2000/10/27Amino Acid SequenceanalysisAnimalsAnkyrin RepeatBrainCalcium SignalingCationsCentral Nervous SystemchemistryChickensCho CellsChromosomes,Human,Pair 12Chromosomes,Human,Pair 17Cloning,MolecularCricetinaecytologyElectrophysiologyGene Expression ProfilingGenesgeneticsHairHair Cells,Auditory,InnerHumanHumansHypotonic SolutionsIn Situ HybridizationIon Channel GatingIon ChannelsMerkel CellsmetabolismMiceMolecular Sequence DataNervous SystemNeuronsNeurons,AfferentOsmolar ConcentrationOsmotic PressurePhylogenyphysiologyProteinsRadiation Hybrid MappingRatsReceptors,DrugResearchRnaRNA,MessengerSequence AlignmentNot in File525535Cell1033Laboratory of Molecular Genetics, The Rockefeller University, New York, New York 10021, USAPM:11081638Cell1Strotmann2000318OTRPC4, a nonselective cation channel that confers sensitivity to extracellular osmolarityJournal318OTRPC4, a nonselective cation channel that confers sensitivity to extracellular osmolarityStrotmann,R.Harteneck,C.Nunnenmacher,K.Schultz,G.Plant,T.D.2000/10Amino Acid SequenceAnimalsCalciumCation Transport ProteinsCationschemistryComparative StudyElectric ConductivityElectrophysiologygeneticsHeartIon Channelsisolation & purificationKidneyLivermetabolismMiceMolecular Sequence DataMyocardiumOsmotic PressurePatch-Clamp TechniquesProteinsSequence Homology,Amino AcidSignal TransductionTissue DistributionTRPV Cation ChannelsNot in File695702Nat.Cell Biol.210Institut fur Pharmakologie, Universitatsklinikum Benjamin Franklin, Freie Universitat Berlin, Thielallee 67-73, 14195 Berlin, GermanyPM:11025659Nat.Cell Biol.1Wissenbach2000320Trp12, a novel Trp related protein from kidneyJournal320Trp12, a novel Trp related protein from kidneyWissenbach,U.Bodding,M.Freichel,M.Flockerzi,V.2000/11/24Amino Acid SequenceanalysisAnimalsCalciumCalcium ChannelsCell LinechemistryCho CellsChromosomes,Human,Pair 12Cloning,MolecularCodonCricetinaeCytosolDnaExonsGene ExpressiongeneticsHumanHumansHypotonic SolutionsIntronsIon ChannelsKidneymetabolismMiceMolecular Sequence DatapharmacologyphysiologyPolymerase Chain ReactionResearchRnaRNA SplicingRNA,MessengerSequence HomologyTransfectionTRPC Cation ChannelsNot in File127134FEBS Lett.4852-3Institut fur Pharmakologie und Toxikologie der Universitat des Saarlandes, D-66421 Homburg, Germany. ulrich.wissenbach@med-rz.uni-saarland.dePM:11094154FEBS Lett.1(21-23), in the lamina terminalis of the mouse brain, in neurons of the arched vascular organ of the lamina terminalis (VOLT), in the median preoptic area, the optic chiasm, neurons of the subfornical organ, the ventral hippocampal commissure, anterior hypothalamic structures, ependymal cells of the choroid plexus in the lateral ventricles, and dorsal root ganglia (DRG) neurons ADDIN REFMGR.CITE Liedtke2000319Vanilloid receptor-related osmotically activated channel (VR-OAC), a candidate vertebrate osmoreceptorJournal319Vanilloid receptor-related osmotically activated channel (VR-OAC), a candidate vertebrate osmoreceptorLiedtke,W.Choe,Y.Marti-Renom,M.A.Bell,A.M.Denis,C.S.Sali,A.Hudspeth,A.J.Friedman,J.M.Heller,S.2000/10/27Amino Acid SequenceanalysisAnimalsAnkyrin RepeatBrainCalcium SignalingCationsCentral Nervous SystemchemistryChickensCho CellsChromosomes,Human,Pair 12Chromosomes,Human,Pair 17Cloning,MolecularCricetinaecytologyElectrophysiologyGene Expression ProfilingGenesgeneticsHairHair Cells,Auditory,InnerHumanHumansHypotonic SolutionsIn Situ HybridizationIon Channel GatingIon ChannelsMerkel CellsmetabolismMiceMolecular Sequence DataNervous SystemNeuronsNeurons,AfferentOsmolar ConcentrationOsmotic PressurePhylogenyphysiologyProteinsRadiation Hybrid MappingRatsReceptors,DrugResearchRnaRNA,MessengerSequence AlignmentNot in File525535Cell1033Laboratory of Molecular Genetics, The Rockefeller University, New York, New York 10021, USAPM:11081638Cell1Strotmann2000318OTRPC4, a nonselective cation channel that confers sensitivity to extracellular osmolarityJournal318OTRPC4, a nonselective cation channel that confers sensitivity to extracellular osmolarityStrotmann,R.Harteneck,C.Nunnenmacher,K.Schultz,G.Plant,T.D.2000/10Amino Acid SequenceAnimalsCalciumCation Transport ProteinsCationschemistryComparative StudyElectric ConductivityElectrophysiologygeneticsHeartIon Channelsisolation & purificationKidneyLivermetabolismMiceMolecular Sequence DataMyocardiumOsmotic PressurePatch-Clamp TechniquesProteinsSequence Homology,Amino AcidSignal TransductionTissue DistributionTRPV Cation ChannelsNot in File695702Nat.Cell Biol.210Institut fur Pharmakologie, Universitatsklinikum Benjamin Franklin, Freie Universitat Berlin, Thielallee 67-73, 14195 Berlin, GermanyPM:11025659Nat.Cell Biol.1Wissenbach2000320Trp12, a novel Trp related protein from kidneyJournal320Trp12, a novel Trp related protein from kidneyWissenbach,U.Bodding,M.Freichel,M.Flockerzi,V.2000/11/24Amino Acid SequenceanalysisAnimalsCalciumCalcium ChannelsCell LinechemistryCho CellsChromosomes,Human,Pair 12Cloning,MolecularCodonCricetinaeCytosolDnaExonsGene ExpressiongeneticsHumanHumansHypotonic SolutionsIntronsIon ChannelsKidneymetabolismMiceMolecular Sequence DatapharmacologyphysiologyPolymerase Chain ReactionResearchRnaRNA SplicingRNA,MessengerSequence HomologyTransfectionTRPC Cation ChannelsNot in File127134FEBS Lett.4852-3Institut fur Pharmakologie und Toxikologie der Universitat des Saarlandes, D-66421 Homburg, Germany. ulrich.wissenbach@med-rz.uni-saarland.dePM:11094154FEBS Lett.1(24-26). The broad spectrum of activators and the wide distribution of TRPV4 suggest that the functions of TRPV4 extend beyond osmosensation.
TRPV4 has been proposed to play a role in the mechanical hyperalgesia that is generated by the concerted action of inflammatory mediators present in inflamed tissues ADDIN REFMGR.CITE Alessandri-Haber2006542A transient receptor potential vanilloid 4-dependent mechanism of hyperalgesia is engaged by concerted action of inflammatory mediatorsJournal542A transient receptor potential vanilloid 4-dependent mechanism of hyperalgesia is engaged by concerted action of inflammatory mediatorsAlessandri-Haber,N.Dina,O.A.Joseph,E.K.Reichling,D.Levine,J.D.2006/4/5AnimalsCarrageenanCells,CulturedGanglia,SpinalHyperalgesiaimmunologyIn VitroInflammationInflammation MediatorsMaleMechanotransduction,CellularMiceNeuronsProtein Kinase CRatsRats,Sprague-DawleyReflexResearchsurgeryTouchTRPV Cation ChannelsNot in File38643874J.Neurosci.2614Division of Neurosciences, Department of Oral and Maxillofacial Surgery, University of California, San Francisco, California 94143-0440, USA. haber@itsa.ucsf.eduPM:16597741J.Neurosci.1(27). After tissue injury, inflammatory mediators such as bradykinin, PGE2, 5-HT and histamine directly sensitize primary afferent neurons, resulting in hyperalgesia ADDIN REFMGR.CITE Huang20063809Modulation of temperature-sensitive TRP channelsreviewed in Journal3809Modulation of temperature-sensitive TRP channelsHuang,J.Zhang,X.McNaughton,P.A.2006/12AACTIVATIONAnimalAnimalsarticlecellcell membraneCHANNELColdheation channelIon ChannelsmembranemodulationOrpharmacologyphosphorylationprotein kinaseProtein KinasesreviewtemperatureNot in File638645Semin.Cell Dev.Biol.176Department of Pharmacology, University of Cambridge, Cambridge CB2 1PD, UKPM:17185012Semin.Cell Dev.Biol.1(reviewed in 28). Important intracellular signaling molecules contributing to inflammatory hyperalgesia include protein kinase C (PKC) ADDIN REFMGR.CITE Cesare199619A novel heat-activated current in nociceptive neurons and its sensitization by bradykininJournal19A novel heat-activated current in nociceptive neurons and its sensitization by bradykininCesare,P.McNaughton,P.1996/12/24AnimalAnimals,Newbornantagonists & inhibitorsBradykininCalciumCationsCells,Cultureddrug effectsEnzyme ActivationEnzyme InhibitorsGanglia,SpinalHeatKineticsMembrane PotentialsmetabolismNeuronsNeurons,AfferentNociceptorsOxazolesPainpharmacologyphysiologyphysiopathologyProtein Kinase CProtein-Tyrosine-PhosphataseRatsRats,WistarStaurosporineSupport,Non-U.S.Gov'tTetradecanoylphorbol AcetateTime FactorsIn File1543515439Proc.Natl.Acad.Sci.U.S.A9326Department of Physiology, King's College London, Strand, United KingdomPM:8986829Proc.Natl.Acad.Sci.U.S.A1Cesare199939Specific involvement of PKC-epsilon in sensitization of the neuronal response to painful heatJournal39Specific involvement of PKC-epsilon in sensitization of the neuronal response to painful heatCesare,P.Dekker,L.V.Sardini,A.Parker,P.J.McNaughton,P.A.1999/7AnimalAnimals,Newbornantagonists & inhibitorsBiological TransportBradykininCarcinogensCell MembraneCells,Culturedcytologydrug effectsEnzyme ActivationenzymologyGanglia,SpinalHeatIsoenzymesmetabolismNeuronsNeurons,AfferentNociceptorsPainPatch-Clamp TechniquespharmacologyphysiologyProtein Kinase CRatsStimulation,ChemicalSupport,Non-U.S.Gov'tTetradecanoylphorbol AcetateIn File617624Neuron233Neuroscience Research Centre, School of Biomedical Sciences, King's College London, Strand, United KingdomPM:10433272Neuron1(29;30) and cyclic AMP-dependent protein kinase (PKA) ADDIN REFMGR.CITE Bhave2002331cAMP-dependent protein kinase regulates desensitization of the capsaicin receptor (VR1) by direct phosphorylationJournal331cAMP-dependent protein kinase regulates desensitization of the capsaicin receptor (VR1) by direct phosphorylationBhave,G.Zhu,W.Wang,H.Brasier,D.J.Oxford,G.S.Gereau,R.W.2002/8/15Amino Acid Sequenceanalogs & derivativesanalysisAnimalsBinding SitesCapsaicinCentral Nervous SystemCho CellsCos CellsCricetinaeCyclic AMPCyclic AMP-Dependent Protein Kinasesdiagnostic usedrug effectsenzymologygeneticsHypersensitivityIn VitroinjuriesmetabolismMutationNeuronsNeurons,AfferentNociceptorsPainpharmacologyPhosphorylationProtein KinasesProtein Structure,TertiaryProteinsReceptors,DrugRecombinant Fusion ProteinsResearchSerineTransfectionNot in File721731Neuron354Division of Neuroscience, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USAPM:12194871Neuron1(31). For example, the activation of the Gq-coupled B1 and B2 receptors by bradykinin leads to the release of a range of potential intracellular messengers, with a substantial body of evidence favouring the idea that the temperature threshold of TRPV1 is lowered by PKC(-mediated phosphorylation ADDIN REFMGR.CITE Cesare199619A novel heat-activated current in nociceptive neurons and its sensitization by bradykininJournal19A novel heat-activated current in nociceptive neurons and its sensitization by bradykininCesare,P.McNaughton,P.1996/12/24AnimalAnimals,Newbornantagonists & inhibitorsBradykininCalciumCationsCells,Cultureddrug effectsEnzyme ActivationEnzyme InhibitorsGanglia,SpinalHeatKineticsMembrane PotentialsmetabolismNeuronsNeurons,AfferentNociceptorsOxazolesPainpharmacologyphysiologyphysiopathologyProtein Kinase CProtein-Tyrosine-PhosphataseRatsRats,WistarStaurosporineSupport,Non-U.S.Gov'tTetradecanoylphorbol AcetateTime FactorsIn File1543515439Proc.Natl.Acad.Sci.U.S.A9326Department of Physiology, King's College London, Strand, United KingdomPM:8986829Proc.Natl.Acad.Sci.U.S.A1Cesare199939Specific involvement of PKC-epsilon in sensitization of the neuronal response to painful heatJournal39Specific involvement of PKC-epsilon in sensitization of the neuronal response to painful heatCesare,P.Dekker,L.V.Sardini,A.Parker,P.J.McNaughton,P.A.1999/7AnimalAnimals,Newbornantagonists & inhibitorsBiological TransportBradykininCarcinogensCell MembraneCells,Culturedcytologydrug effectsEnzyme ActivationenzymologyGanglia,SpinalHeatIsoenzymesmetabolismNeuronsNeurons,AfferentNociceptorsPainPatch-Clamp TechniquespharmacologyphysiologyProtein Kinase CRatsStimulation,ChemicalSupport,Non-U.S.Gov'tTetradecanoylphorbol AcetateIn File617624Neuron233Neuroscience Research Centre, School of Biomedical Sciences, King's College London, Strand, United KingdomPM:10433272Neuron1Numazaki2002297Direct phosphorylation of capsaicin receptor VR1 by PKCe and identification of two target serine residuesJournal297Direct phosphorylation of capsaicin receptor VR1 by PKCe and identification of two target serine residuesNumazaki,M.Tominaga,T.Toyooka,H.Tominaga,M.2002/3/7analysisCapsaicinIn VitroInjection of anti-NGF reduces thermal hyperalgesiaPainPhosphorylationphysiologyProteinsProtonsSerineTemperatureIn FileJ Biol ChemDept. of Physiology, Mie Univ. School of Medicine, Tsu, Mie 514-8507PM:11884385J Biol Chem1Bhave2003330Protein kinase C phosphorylation sensitizes but does not activate the capsaicin receptor transient receptor potential vanilloid 1 (TRPV1)Journal330Protein kinase C phosphorylation sensitizes but does not activate the capsaicin receptor transient receptor potential vanilloid 1 (TRPV1)Bhave,G.Hu,H.J.Glauner,K.S.Zhu,W.Wang,H.Brasier,D.J.Oxford,G.S.Gereau,R.W.2003/10/14AlanineAnimalsCapsaicinchemistryCos Cellsdrug effectsEnzyme ActivationgeneticsIn VitroIon Channel GatingKineticsLigandsmetabolismMutationpharmacologyPhosphorylationProtein Kinase CProteinsReceptors,DrugRecombinant Fusion ProteinsResearchTetradecanoylphorbol AcetateTransfectionNot in File1248012485Proc.Natl.Acad.Sci.U.S.A10021Division of Neuroscience, Baylor College of Medicine, Houston, TX 77030, USAPM:14523239Proc.Natl.Acad.Sci.U.S.A1(32-35). PKA, like PKC, is a critical intracellular signalling molecule mediating inflammatory hyperalgesia ADDIN REFMGR.CITE Levine1999416Peripheral mechanisms of inflammatory pain. In: Wall PD, Melzack R, editors. Textbook of pain. 4th ed. London (U.K.)Book Chapter416Peripheral mechanisms of inflammatory pain. In: Wall PD, Melzack R, editors. Textbook of pain. 4th ed. London (U.K.)Levine,J.D.Reichling,D.B.1999PainNot in File59843(36). In sensory neurons PGE2 activates both the EP1 receptor, which is Gq-coupled and therefore activates PKC, and the EP4 receptor, which is Gs-coupled and therefore activates PKA. Cyclic AMP analogues, the adenylate cyclase activator, forskolin (FSK), or phosphodiesterase inhibitors enhance the mechanical and thermal hyperalgesic effects of PGE2 ADDIN REFMGR.CITE Cunha1999550Pharmacological modulation of secondary mediator systems--cyclic AMP and cyclic GMP--on inflammatory hyperalgesiaJournal550Pharmacological modulation of secondary mediator systems--cyclic AMP and cyclic GMP--on inflammatory hyperalgesiaCunha,F.Q.Teixeira,M.M.Ferreira,S.H.1999/6AnimalsBradykininCarrageenanchemically inducedCyclic AMPCyclic GMPCytokinesDinoprostoneDopaminedrug effectsHyperalgesiaInflammationInterleukin-1Interleukin-6Interleukin-8IsoquinolinesMalemetabolismOxadiazolespathologypharmacologyPhosphodiesterase InhibitorsphysiologyphysiopathologyPyrrolidinonesQuinoxalinesRatsRats,WistarResearchRolipramSecond Messenger SystemsSulfonamidesTumor Necrosis Factor-alphaNot in File671678Br.J.Pharmacol.1273Department of Pharmacology, Faculty of Medicine of Ribeirao Preto, University of Sao Paulo, BrazilPM:10401557Br.J.Pharmacol.1Taiwo1989548Mediation of primary afferent peripheral hyperalgesia by the cAMP second messenger systemJournal548Mediation of primary afferent peripheral hyperalgesia by the cAMP second messenger systemTaiwo,Y.O.Bjerknes,L.K.Goetzl,E.J.Levine,J.D.19898-Bromo Cyclic Adenosine MonophosphateAdenosineAdenosine MonophosphateAnimalsArachidonic Acidchemically inducedCyclic AMPdrug effectsEicosanoidsHyperalgesiaHyperesthesiaIndomethacinMalemetabolismNociceptorsPeripheral NervespharmacologyphysiologyphysiopathologyRatsRats,Inbred StrainsResearchSecond Messenger SystemsNot in File577580Neuroscience323Department of Medicine, University of California 94143-0724PM:2557557Neuroscience1Taiwo1991549Further confirmation of the role of adenyl cyclase and of cAMP-dependent protein kinase in primary afferent hyperalgesiaJournal549Further confirmation of the role of adenyl cyclase and of cAMP-dependent protein kinase in primary afferent hyperalgesiaTaiwo,Y.O.Levine,J.D.19911-Methyl-3-isobutylxanthineAdenosineAdenosine MonophosphateAdenylate Cyclaseanalogs & derivativesAnimalschemically inducedCyclic AMPDinoprostonedrug effectsenzymologyEpoprostenolForskolinHyperalgesiaMaleNociceptorsPhosphodiesterase InhibitorsPhosphorylationphysiologyphysiopathologyProtein Kinase CProtein KinasesPyrrolidinonesRatsRats,Inbred StrainsResearchRolipramSecond Messenger SystemsSensory ThresholdsStaurosporineThionucleotidestoxicityNot in File131135Neuroscience441Department of Medicine, University of California, San Francisco 94143-0724PM:1722888Neuroscience1(37-39). Thus PKC and PKA have a vital role to play in the process of inflammatory hyperalgesia.
The speed and specificity of the action of kinases is in many cases enhanced by binding to scaffolding proteins, which pre-assemble the kinases into signalling complexes with their target substrates. The AKAP (A Kinase Anchoring Protein) family of scaffolding proteins was originally named for their ability to target PKA to appropriate substrates, but are now known to assemble a wide range of kinases and phosphatases into signalling complexes with appropriate targets ADDIN REFMGR.CITE Smith2006400The where's and when's of kinase anchoringJournal400The where's and when's of kinase anchoringSmith,F.D.Langeberg,L.K.Scott,J.D.2006/6Adaptor Proteins,Signal TransducingAnimalsCyclic AMPCyclic AMP-Dependent Protein KinasesHumansmetabolismPhosphorylationphysiologyProtein KinasesProtein Processing,Post-TranslationalProteinsResearchSignal TransductionNot in File316323Trends Biochem.Sci.316Howard Hughes Medical Institute, Vollum Institute, Oregon Health and Sciences University, MRB322, 3181 SW Sam Jackson Park Road, Portland, OR 97239, USAPM:16690317Trends Biochem.Sci.1(40). A number of ion channels are subject to modulation by AKAPs, including glutamate receptors, calcium channels and the M-type potassium channels ADDIN REFMGR.CITE Altier2002401Trafficking of L-type calcium channels mediated by the postsynaptic scaffolding protein AKAP79Journal401Trafficking of L-type calcium channels mediated by the postsynaptic scaffolding protein AKAP79Altier,C.Dubel,S.J.Barrere,C.Jarvis,S.E.Stotz,S.C.Spaetgens,R.L.Scott,J.D.Cornet,V.De,Waard M.Zamponi,G.W.Nargeot,J.Bourinet,E.2002/9/13A Kinase Anchor ProteinsAdaptor Proteins,Signal TransducingAmino Acid MotifsAmino Acid SequenceanalysisBiological TransportCalciumCalcium ChannelsCalcium Channels,L-TypeCalcium SignalingCarrier ProteinschemistryHemagglutininsHumanHumansmetabolismMolecular Sequence DataPhosphorylationphysiologyProteinsResearchSignal TransductionNot in File3359833603J.Biol.Chem.27737Physiopathologie des Canaux Ioniques, Institut de Genetique Humaine CNRS UPR1142, 141 Rue de la Cardonille, 34396 Montpellier Cedex 5, FrancePM:12114507J.Biol.Chem.1Colledge2000402Targeting of PKA to glutamate receptors through a MAGUK-AKAP complexJournal402Targeting of PKA to glutamate receptors through a MAGUK-AKAP complexColledge,M.Dean,R.A.Scott,G.K.Langeberg,L.K.Huganir,R.L.Scott,J.D.2000/7Adaptor Proteins,Signal TransducingAnimalsBinding,CompetitiveBlotting,WesternCarrier ProteinsCyclic AMP-Dependent Protein KinasescytologyGuanylate KinaseHippocampusImmunohistochemistryIntracellular Signaling Peptides and ProteinsMembrane ProteinsmetabolismMicroscopy,ConfocalNerve Tissue ProteinsNeuronsNucleoside-Phosphate KinasePeptidesPhosphorylationphysiologyPrecipitin TestsProtein BindingProtein KinasesProteinsRatsReceptors,AMPAReceptors,GlutamateReceptors,N-Methyl-D-AspartateResearchSignal TransductionSynaptic TransmissionNot in File107119Neuron271Howard Hughes Medical Institute, Vollum Institute, Oregon Health Sciences University, Portland 97201, USAPM:10939335Neuron1Hoshi2005403Distinct enzyme combinations in AKAP signalling complexes permit functional diversityJournal403Distinct enzyme combinations in AKAP signalling complexes permit functional diversityHoshi,N.Langeberg,L.K.Scott,J.D.2005/11Adaptor Proteins,Signal Transducingagonistsalpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acidantagonists & inhibitorsCalcineurinCarrier ProteinsCell LineCellsCyclic AMP-Dependent Protein KinasesHumanHumansIon ChannelsMembrane ProteinsMembrane Transport ProteinsmetabolismModels,BiologicalMuscarinic AgonistsphysiologyPotassiumPotassium ChannelsProtein Kinase CProtein KinasesProteinsResearchRnaRNA InterferenceSignal TransductionTime FactorsTransfectionNot in File10661073Nat.Cell Biol.711Howard Hughes Medical Institute, Vollum Institute, Oregon Health and Science University, 3181 S.W. Sam Jackson Park Road, Portland, OR 97239, USAPM:16228013Nat.Cell Biol.1Hoshi2003404AKAP150 signaling complex promotes suppression of the M-current by muscarinic agonistsJournal404AKAP150 signaling complex promotes suppression of the M-current by muscarinic agonistsHoshi,N.Zhang,J.S.Omaki,M.Takeuchi,T.Yokoyama,S.Wanaverbecq,N.Langeberg,L.K.Yoneda,Y.Scott,J.D.Brown,D.A.Higashida,H.2003/6A Kinase Anchor ProteinsAdaptor Proteins,Signal TransducingagonistsanalysisAnimalsantagonists & inhibitorsCarrier ProteinsCell MembraneCells,CulturedDiglyceridesdrug effectsEnzyme InhibitorsGanglia,SympatheticgeneticsKCNQ2 Potassium ChannelMacromolecular SubstancesMembrane PotentialsmetabolismMuscarinic AgonistsMutationNeural InhibitionNeuronsPeptide FragmentsPeptidespharmacologyPhosphorylationphysiologyPotassiumPotassium ChannelsPotassium Channels,Voltage-GatedProtein BindingProtein IsoformsProtein Kinase CProteinsRatsResearchSignal TransductionNot in File564571Nat.Neurosci.66Department of Biophysical Genetics, Kanazawa University Graduate School of Medicine, 13-1 Takara-machi, Kanazawa, Ishikawa 920-8640, Japan. hoshin@ohsu.eduPM:12754513Nat.Neurosci.1(41-44). The heat-activated ion channel TRPV1, a member of the same subfamily as TRPV4, has recently been shown to be assembled into a signaling complex with PKA, PKC, and PP2B by AKAP79, and the sensitization of TRPV1 by PKC and PKA is critically reliant on binding to AKAP79 ADDIN REFMGR.CITE Zhang20084072Proinflammatory mediators modulate the heat-activated ion channel TRPV1 via the scaffolding protein AKAP79/150Journal4072Proinflammatory mediators modulate the heat-activated ion channel TRPV1 via the scaffolding protein AKAP79/150Zhang,X.Li,L.McNaughton,P.A.2008/8/14AACTIVATIONarticleAsAtbradykininCalcineurinCHANNELheathyperalgesiainflammationinjuriesion channelIon ChannelsmediatormodulationOnOrpharmacologyphosphataseprotein kinaseProtein KinasesResearchsensitizationtemperaturevertebrateVertebratesNot in File450461Neuron593Department of Pharmacology, University of Cambridge, Cambridge CB21PD, UKPM:18701070http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6WSS-4T6MHFJ-F&_user=1495569&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_version=1&_urlVersion=0&_userid=1495569&md5=bbb9076712295e11114aad2290246beeNeuron1(45).
The present study shows that PKC and PKA activation can sensitize TRPV4 to mechanical stimuli, identifies the relevant phosphorylation sites, and shows that the scaffolding protein AKAP79 plays a critical role in sensitization of TRPV4.
Experimental procedures
Cell culture and transfection. HEK293 cel l s ( A T C C ) w e r e m a i n t a i n e d i n D M E M s u p p l e m e n t e d w i t h 1 0 % F B S , 5 0 U / m l p e n i c i l l i n , 5 0 g / m l s t r e p t o m y c i n a n d 2 0 m M L - g l u t a m i n e . C e l l s w e r e t r a n s i e n t l y t r a n s f e c t e d u s i n g P o l y F e c t r e a g e n t ( Q i a g e n ) i n a c c o r d a n c e w i t h t h e m a n u f a c t u r e r s i n s t r u c t i o n s w i t h s o m e m o d i f i c a t i o n a s f o l l o w s . B r i e f l y , 2 g D N A w a s d i l u t e d i n a t o t a l v o l u m e o f 1 0 0 l s e r u m f r e e m e d i u m a n d m i x e d w i t h 1 0 l P o l y F e c t t r a n s f e c t i o n r e a g e n t , f o l l o w e d b y 1 0 m i n u t e s r o o m t e m p e r a t u r e i n c u b a t i o n t o a l l o w D N A c o m p l e x f o r m a t i o n , 0 . 6 m l o f g r o w t h m e d i u m w a s added to the transfection complexes, DNA solution was transferred to the cells and was incubated for 24 hours.
Plasmid and DNA construction. Potential TRPV4 PKC and PKA phosphorylation sites were sought by using GPS ( HYPERLINK "http://973-proteinweb.ustc.edu.cn/gps/gps_web/predict.php" http://973-proteinweb.ustc.edu.cn/gps/gps_web/predict.php) ADDIN REFMGR.CITE Zhou2004480GPS: a novel group-based phosphorylation predicting and scoring methodJournal480GPS: a novel group-based phosphorylation predicting and scoring methodZhou,F.F.Xue,Y.Chen,G.L.Yao,X.2004/12/24AcidsAlgorithmsAmino AcidsBinding SiteschemistryclassificationComparative StudyConserved SequencemethodsPhosphorylationProtein BindingProtein KinasesProtein-Serine-Threonine KinasesProteinsResearchSequence AlignmentSequence Analysis,ProteinSequence Homology,Amino AcidSerineThreonineValineNot in File14431448Biochem.Biophys.Res.Commun.3254Department of Computer Science and Technology, University of Science and Technology of China, Hefei, Anhui 230027, PR ChinaPM:15555589Biochem.Biophys.Res.Commun.1(46), PredPhospho ( HYPERLINK "http://www.pred.ngri.re.kr/PredPhospho.htm" www.pred.ngri.re.kr/PredPhospho.htm) ADDIN REFMGR.CITE Kim2004486Prediction of phosphorylation sites using SVMsJournal486Prediction of phosphorylation sites using SVMsKim,J.H.Lee,J.Oh,B.Kimm,K.Koh,I.2004/11/22AlgorithmsArtificial IntelligenceBinding SiteschemistryComparative StudyComputer SimulationGenomemethodsModels,ChemicalModels,MolecularPhosphorylationPhosphotransferasesProteinsResearchSequence AlignmentSequence Analysis,ProteinSignal TransductionStructure-Activity RelationshipNot in File31793184Bioinformatics.2017National Genome Research Institute, 5 Nokbun-Dong, Eunpyung-Gu, Seoul, 122-701 Korea. jh7521@ngri.re.krPM:15231530Bioinformatics.1(47), NetPhosK ( HYPERLINK "http://www.cbs.dtu.dk/services/NetPhosK" www.cbs.dtu.dk/services/NetPhosK) ADDIN REFMGR.CITE Blom2004484Prediction of post-translational glycosylation and phosphorylation of proteins from the amino acid sequenceJournal484Prediction of post-translational glycosylation and phosphorylation of proteins from the amino acid sequenceBlom,N.Sicheritz-Ponten,T.Gupta,R.Gammeltoft,S.Brunak,S.2004/6Amino Acid SequenceanalysisAnimalschemistrygeneticsGlycosylationHumansInternetmetabolismmethodsNeural Networks (Computer)PhosphorylationProtein KinasesProtein Processing,Post-TranslationalProteinsReproducibility of ResultsSensitivity and SpecificityNot in File16331649Proteomics.46Center for Biological Sequence Analysis, The Technical University of Denmark, Lyngby, Denmark. nikob@cbs.dtu.dkPM:15174133Proteomics.1(48), and ScanSite ( HYPERLINK "http://www.scansite.mit.edu" www.scansite.mit.edu) ADDIN REFMGR.CITE Yaffe2001485A motif-based profile scanning approach for genome-wide prediction of signaling pathwaysJournal485A motif-based profile scanning approach for genome-wide prediction of signaling pathwaysYaffe,M.B.Leparc,G.G.Lai,J.Obata,T.Volinia,S.Cantley,L.C.2001/4AlgorithmsAmino Acid MotifsAmino Acid SequenceAnimalsCattlechemistryDatabases,FactualGenomeHumanHumansInternetMiceMolecular Sequence DataProtein KinasesProteinsRatsResearchSerineSignal TransductionSoftwareThreonineTyrosineNot in File348353Nat.Biotechnol.194Division of Signal Transduction, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Institutes of Medicine, 10th Floor, 330 Brookline Ave. Boston, MA 02215 USA. myaffe@mit.eduPM:11283593Nat.Biotechnol.1(49). Potential phosphorylation sites were targeted when they were identified with high probability by one or more of these packages.
The siRNA against AKAP79 was constructed in a plasmid co-expressing GFP to facilitate identification of successfully transfected cells, as previously described ADDIN REFMGR.CITE Zhang20084072Proinflammatory mediators modulate the heat-activated ion channel TRPV1 via the scaffolding protein AKAP79/150Journal4072Proinflammatory mediators modulate the heat-activated ion channel TRPV1 via the scaffolding protein AKAP79/150Zhang,X.Li,L.McNaughton,P.A.2008/8/14AACTIVATIONarticleAsAtbradykininCalcineurinCHANNELheathyperalgesiainflammationinjuriesion channelIon ChannelsmediatormodulationOnOrpharmacologyphosphataseprotein kinaseProtein KinasesResearchsensitizationtemperaturevertebrateVertebratesNot in File450461Neuron593Department of Pharmacology, University of Cambridge, Cambridge CB21PD, UKPM:18701070http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6WSS-4T6MHFJ-F&_user=1495569&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_version=1&_urlVersion=0&_userid=1495569&md5=bbb9076712295e11114aad2290246beeNeuron1(45). We thank the following for kind gifts of plasmids: hTRPV4 tagged with V5, Dr David Cohen; AKAP79, Dr John Scott; hB2 receptor, Dr Tanya ONeill.
Point mutations were introduced into TRPV4 by using QuikChange (Stratagene) site directed mutagenesis in accordance with the manufacturers instructions. The S189A/T175A double mutant was constructed by combination of the S189A site mutant together with the T175A mutant. The S162A/S189A/T175A triple mutant was constructed by combination of the S162A site mutant together with the double mutant S189A/T175A. All constructs were verified by DNA sequencing (Department of Biochemistry, University of Cambridge). The cDNAs were subcloned into the pcDNA3 vector (Invitrogen) for amplification and transfection.
Imaging of intracellular calcium and data analysis. Calcium imaging was performed as described previously (Bonnington et al., 2003). Briefly, HEK293 cells were transiently transfected with 2 g T R P V 4 c D N A ( W T o r m u t a n t ) , o r c o t r a n s f e c t e d w i t h 0 . 6 7 g w t T R P V 4 c D N A + 1 . 3 3 g A K A P 7 9 o r s i R N A A K A P 7 9 f o r 2 4 h o u r s . C e l l s w e r e p l a t e d o n t o 1 3 m m p o l y l y s i n e - t r e a t e d c o v e r s l i p s a n d g r o w n f o r o n e d a y , l o a d e d w i t h 1 0 M f l u o - 4 A M ( M o l e c u l a r P r o b e s ) f o r 4 0 m i n utes, and were then mounted into a chamber which was continuously perfused with an isotonic solution (100 mM NaCl, 2 mM CaCl2, 4mM KCl, 1 mM MgCl2, 10 mM HEPES, 10 mM glucose, 100 mM Mannitol, pH 7.4, osmolarity 320 5 milliosm). [Ca2+]i was monitored with an inverted confocal microscope (BioRad). Cell swelling was induced by omitting mannitol from this solution (230 5 milliosm). Fluo-4 AM was excited at 488 nm, and images of the fluorescence, F, were captured every 3 seconds. At the end of the experiment, the maximal fluorescence (Fmax) was obtained by application of the calcium ionophore ionomycin (10 M). The Ca2+-dependent fluorescence changes were calibrated as described ADDIN REFMGR.CITE Zhang2005398NGF rapidly increases membrane expression of TRPV1 heat-gated ion channelsJournal398NGF rapidly increases membrane expression of TRPV1 heat-gated ion channelsZhang,X.Huang,J.McNaughton,P.A.2005/12/211-Phosphatidylinositol 3-KinaseAmino Acid SequenceAnimalsBradykininCalciumCapsaicinCell LineCells,CulturedchemistryDnaDNA,ComplementaryElectrophysiologyGene Expression RegulationGlutathione TransferaseHeatHumanHumansHyperalgesiaImmunoblottingImmunohistochemistryIon ChannelsmetabolismMiceModels,BiologicalMolecular Sequence DataMutationNerve Growth FactorNeuronsNociceptorspharmacologyPhosphorylationphysiologyPlasmidsProtein BindingProteinsReceptor,trkARecombinant Fusion ProteinsResearchSequence Homology,Amino AcidSignal TransductionSodiumsrc-Family KinasesTime FactorsTransfectionTRPV Cation ChannelsTyrosineVanadatesNot in File42114223EMBO J.2424Department of Pharmacology, University of Cambridge, Cambridge, UKPM:16319926EMBO J.1(50). Data are expressed as F/Fmax. Inhibitors were applied for 30 minutes before treatment with e.g. PMA, FSK or bradykinin. Significance was tested using one-way analysis of variance (ANOVA) with Bonferronis post hoc test (SPSS for Windows). Significance levels in figures are given as: ns, not significant, >5%; *, <5%; **, <1%; ***, <0.1%. All errors shown as bars in Figures or quoted in the text are SEM. All experiments were performed at room temperature (2022C).
Immunoprecipitation and immunoblotting. The following antibodies were used: anti-phosphoserine antibody (PSR-45, Sigma), anti-V5 (Invitrogen). Following treatment as specified in figure legends, transfected cells were solubilized in lysis buffer (20mM Tris, pH 8.0, 150mM NaCl, 1mM EDTA, 2mM EGTA, 1%NP-40, 1mM PMSF, 50mM NaF, 10% protease inhibitors (Roche), and 1x phosphatase inhibitor (Sigma)), cell ly s a t e s w e r e c e n t r i f u g e d a t 1 2 , 0 0 0 r p m f o r 1 0 m i n u t e s , c l e a r e d s u p e r n a t a n t w a s m i x e d w i t h 1 l m o u s e a n t i - V 5 , t o p r e c i p i t a t e T R P V 4 , a n d 3 0 l p r o t e i n A - a g a r o s e ( S a n t a C r u z ) , a n d w a s i n c u b a t e d f o r t h r e e h o u r s . I m m u n o c o m p l e x e s w e r e c o l l e c t e d b y c e n t r i f u g a t i o n , a nd the immunoprecipitates were washed three times with lysis buffer followed by boiling for 5 minutes in SDS-PAGE sample buffer. Agarose beads were removed by centrifugation prior to loading the samples onto 7.5% polyacrylamide gels. Proteins were transferred from the resolved SDS-PAGE gels to Hybond-P membrane. Blots were blocked in blocking buffer (PBS containing 0.1% Tween-20 and 1.5% gelatin) at room temperature for one hour and then incubated with diluted primary antibody (PSR-45 1:1000, anti-V5: 1:10,000 in PBS) at 4C for three hours. The blots were then washed in the washing buffer (PBS with 0.1%Tween-20) prior to addition of HRP-conjugated sheep anti-mouse (for PSR-45 and V5) for 1 hour at room temperature. Blots were washed repeatedly and developed using ECL chemiluminescent reagent (Amersham Biosciences) before exposure to X-ray film. All bands quantified with NIH image 1.62 software. As the expression levels of TRPV4 were somewhat variable (see lower blots in Fig. 2A and 5A) the phosphoserine band densities were expressed relative to the relevant TRPV4 band densities. All blots shown in Figures are typical of at least three similar results.
Results
TRPV4 sensitization by PKC activation. We used HEK293 cells transfected with plasmids containing cDNAs coding for TRPV4 to investigate the sensitization of TRPV4. Hypotonic solutions were used to activate TRPV4 ADDIN REFMGR.CITE Liedtke2000319Vanilloid receptor-related osmotically activated channel (VR-OAC), a candidate vertebrate osmoreceptorJournal319Vanilloid receptor-related osmotically activated channel (VR-OAC), a candidate vertebrate osmoreceptorLiedtke,W.Choe,Y.Marti-Renom,M.A.Bell,A.M.Denis,C.S.Sali,A.Hudspeth,A.J.Friedman,J.M.Heller,S.2000/10/27Amino Acid SequenceanalysisAnimalsAnkyrin RepeatBrainCalcium SignalingCationsCentral Nervous SystemchemistryChickensCho CellsChromosomes,Human,Pair 12Chromosomes,Human,Pair 17Cloning,MolecularCricetinaecytologyElectrophysiologyGene Expression ProfilingGenesgeneticsHairHair Cells,Auditory,InnerHumanHumansHypotonic SolutionsIn Situ HybridizationIon Channel GatingIon ChannelsMerkel CellsmetabolismMiceMolecular Sequence DataNervous SystemNeuronsNeurons,AfferentOsmolar ConcentrationOsmotic PressurePhylogenyphysiologyProteinsRadiation Hybrid MappingRatsReceptors,DrugResearchRnaRNA,MessengerSequence AlignmentNot in File525535Cell1033Laboratory of Molecular Genetics, The Rockefeller University, New York, New York 10021, USAPM:11081638Cell1Strotmann2000318OTRPC4, a nonselective cation channel that confers sensitivity to extracellular osmolarityJournal318OTRPC4, a nonselective cation channel that confers sensitivity to extracellular osmolarityStrotmann,R.Harteneck,C.Nunnenmacher,K.Schultz,G.Plant,T.D.2000/10Amino Acid SequenceAnimalsCalciumCation Transport ProteinsCationschemistryComparative StudyElectric ConductivityElectrophysiologygeneticsHeartIon Channelsisolation & purificationKidneyLivermetabolismMiceMolecular Sequence DataMyocardiumOsmotic PressurePatch-Clamp TechniquesProteinsSequence Homology,Amino AcidSignal TransductionTissue DistributionTRPV Cation ChannelsNot in File695702Nat.Cell Biol.210Institut fur Pharmakologie, Universitatsklinikum Benjamin Franklin, Freie Universitat Berlin, Thielallee 67-73, 14195 Berlin, GermanyPM:11025659Nat.Cell Biol.1Wissenbach2000320Trp12, a novel Trp related protein from kidneyJournal320Trp12, a novel Trp related protein from kidneyWissenbach,U.Bodding,M.Freichel,M.Flockerzi,V.2000/11/24Amino Acid SequenceanalysisAnimalsCalciumCalcium ChannelsCell LinechemistryCho CellsChromosomes,Human,Pair 12Cloning,MolecularCodonCricetinaeCytosolDnaExonsGene ExpressiongeneticsHumanHumansHypotonic SolutionsIntronsIon ChannelsKidneymetabolismMiceMolecular Sequence DatapharmacologyphysiologyPolymerase Chain ReactionResearchRnaRNA SplicingRNA,MessengerSequence HomologyTransfectionTRPC Cation ChannelsNot in File127134FEBS Lett.4852-3Institut fur Pharmakologie und Toxikologie der Universitat des Saarlandes, D-66421 Homburg, Germany. ulrich.wissenbach@med-rz.uni-saarland.dePM:11094154FEBS Lett.1(51-53). The calcium influx through the TRPV4 channel, whether activated by heat or by hypotonic solutions, is sensitized by inflammatory mediators in a similar manner ADDIN REFMGR.CITE Alessandri-Haber2006542A transient receptor potential vanilloid 4-dependent mechanism of hyperalgesia is engaged by concerted action of inflammatory mediatorsJournal542A transient receptor potential vanilloid 4-dependent mechanism of hyperalgesia is engaged by concerted action of inflammatory mediatorsAlessandri-Haber,N.Dina,O.A.Joseph,E.K.Reichling,D.Levine,J.D.2006/4/5AnimalsCarrageenanCells,CulturedGanglia,SpinalHyperalgesiaimmunologyIn VitroInflammationInflammation MediatorsMaleMechanotransduction,CellularMiceNeuronsProtein Kinase CRatsRats,Sprague-DawleyReflexResearchsurgeryTouchTRPV Cation ChannelsNot in File38643874J.Neurosci.2614Division of Neurosciences, Department of Oral and Maxillofacial Surgery, University of California, San Francisco, California 94143-0440, USA. haber@itsa.ucsf.eduPM:16597741J.Neurosci.1Gao2003368Temperature-modulated diversity of TRPV4 channel gating: activation by physical stresses and phorbol ester derivatives through protein kinase C-dependent and -independent pathwaysJournal368Temperature-modulated diversity of TRPV4 channel gating: activation by physical stresses and phorbol ester derivatives through protein kinase C-dependent and -independent pathwaysGao,X.Wu,L.O'neil,R.G.2003/7/18analysisAnimalsCalciumCation Transport ProteinsCell LineCho CellsCloning,MolecularCyclic AMP-Dependent Protein Kinasesdrug effectsFura-2geneticsHamstersHumanHumansIon Channel GatingIon ChannelsKidneymetabolismMicePatch-Clamp TechniquespharmacologyPhorbol EstersProtein KinasesProteinsRecombinant ProteinsResearch Support,Non-U.S.Gov'tResearch Support,U.S.Gov't,P.H.S.Ruthenium RedSignal TransductionStaurosporineTemperatureTetradecanoylphorbol AcetateTransfectionNot in File2712927137J.Biol.Chem.27829Department of Integrative Biology and Pharmacology, The University of Texas Health Science Center at Houston, Houston, Texas 77030, USAPM:12738791J.Biol.Chem.1