醫(yī)學(xué)神經(jīng)生物學(xué)：04 Excitatory Amino Acids

ExcitatoryAminoAcidsGlutamate20AminoAcidsUsedfor

ProteinSynthesisNon-essential(Ourbodiescanmakethem)AlanineArginineAsparagineAspartateCysteineGlutamateGlycineGlutamineProlineSerineTyrosineEssential(bodycannotmakethem–mustgetfromdiet)HistidineIsoleucineLeucineLysineMethioninePhenylalanineThreonineTryptophanValineFluxofionsthroughthechannelsispassive

IntracellularExtracellularNa+Na+Cl-Cl-Ca++Ca++K+K+ATPNa+K+Openingandclosingofchannelsrequiresconformationalchange

CLOSEDiontransmitterCLOSEDLigand-gatedionchannelsChannelsactivatedbybindingofaligand:AcetylcholinereceptorsSerotoninreceptorsGABAAreceptorsGlycinereceptorsGlutamatereceptors

pentamerictetramericSubunitStructureGroupIGABAAGlycine5-HTNicotinicSubunitStructureGroupII

AMPAKainateNMDAAminoAcidNTsHighconcentrationinbrain(micromolar)SmallvesiclesPoint-to-pointcommunicationSensory-motorfunctionsConsistentlyexcitatoryorinhibitoryMainlyionotropicreceptorsFastacting,shortduration(1-5ms)LifeCycleofglutamateBiosynthesis&StorageReleaseReceptorActionInactivation2.BiologicalfunctionsofglutamatePhysiologyPathologyGlutamatePrincipalexcitatoryNTBiosynthesizedasbyproductofcellmetabolism(Krebscycle)Removedbyreuptake4receptortypesNMDAAMPAKainatemGluRsMetabotropic

IonotropicAminoAcidsFormedFrom

a-Ketoglutarate(酮戊二酸)TransaminationorGlutamatedehydrogenasea-Keto-glutarateGlutamateGlutamineGlutaminesynthaseGlutaminasePost-synapticNeuronePre-synapticNeuroneiGluRTheglutamate:glutamineshuttle

EAATGliaGlnEPSPGluGluGluYVesiclelumenH+ATPADP+PiYTvYTcVesiculartransportFUNCTIONAccumulationinthepresynapticvesiclesMECHANISMTwosynapticvesicleproteinsmediatetheuptakeoftransmitter:Avacuolarprotonpump;AfamilyoftransmittertransportersCytosolictransportTwofacesofVGLUTs磷酸鹽谷氨酸VGLUT1-3NATUREREVIEWS|NEUROSCIENCE2011AMPA-RPostsynapticmechanismsIonotropicreceptors(GluR)Ca2+NMDA-RNa+Na+Kainate-RMetabotropicreceptors(mGluR)GModulationofintracellularsignaltransductionOpeningofcationchannelsYProcessesinvolvedintheinactivationofsynaptictransmissionYYYYYYUU=uptakeDD=diffusionYZMM=metaboliteYYYYRR=receptordesensitisationOutInCarrier(EAAT)K+CGlu2Na+(EAAT)=Excitatoryaminoacidtransporters

Inactivation

EngineATPNa+K+Drivingforces

K+Na+

GlutamateuptakeGLUCytosol10mMVesicles100mMExtracellular1MTGLUGLUSynapticspace2mM?GlutamateconcentrationgradientsGLUPresynapticPostsynapticGLUGliaK+Na+Ca2+NMDAAMPAKainateIonotropicMetabotropicGGGlutamate-mediatedsynaptictransmissionSUMMARYGLUTGLUGLUGGlutamateisaneurotransmitter4Bindingtoreceptors5Functionaleffects3Uptakenacrossthecellularmembrane1Concentratedinvesicles2ReleasedbyexocytosisASPTASPASPGIsaspartateaneurotransmitter?4Bindingtoreceptors5Functionaleffects3Uptakenacrossthecellularmembrane1Concentratedinvesicles?2Releasedbyexocytosis?Sialinisavesicularaspartate/glutamatetransporterPNAS2008vol.10511721Proteoliposomes:脂蛋白體ATPCl離子Glutamateactson:IonotropicreceptorsMetabotropicreceptorsNMDA-receptorfamilyKainate-receptorfamilyAMPA-receptorfamilyGroupII(Adenylylcyclaseinhibition)GroupIII(Adenylylcyclaseinhibition)GroupI(PhospholipaseCactivation)GluN1GluN2A,B,C,DReceptorsubunitsGluA1…GluA4GluK1…GluK3GluK4GluK5mGlu1mGlu5mGlu2mGlu3mGlu4,mGlu8mGlu6,mGlu7PharmacologyofionotropicglutamatereceptorsGlutamatereceptortypeAgonistsAntagonistsAMPAGlutamate,Quisqualate,AMPA(α-amino-5-methyl-3-hydoxy-4-isoxazolepropionicacid)CNQXNBQXKainateGlutamateKainateCNQXNBQXNMDAGlutamate(alsoglycineisaco-agonistatadifferentbindingsite)NMDA(N-methyl-D-aspartate)AP5(competitive)MK801(ionchannel)Dichlorokynurenicacid(atglycinesite)AMPAreceptorsMolecularstructureofAMPAreceptorsAMPAreceptor-mediatedexcitatorypostsynapticpotential(EPSPs)

majorexcitatoryneurotransmitterinCNSaround70%ofallCNSsynapsesareglutamatergicGluR1-4subunitstructureCOOHNH2insideoutsideTM1TM3TM4TM2

LongextracellularaminoterminusShorterintracellularCterminusThreetransmembranespanningdomains(TM1,TM3andTM4)TM2formsare-entryloopwhichlinesthechannelporeAMPAreceptorsaretetrameric(ie4GluRsubunitscombinetoformreceptor)AMPAreceptorsubunitisoformsandsubunitchannelassembliesIMPERMEABILITYOFAMPARECEPTORSTOCALCIUMGENERATEDBYRNAEDITING（編輯）谷氨酰胺CAG(Q)精氨酸CGG(R)ExpressionofAMPAreceptorsubunitsintheratbrainInsituhybridizationinhorizontalbrainsectionsofadultratsDistributionofAMPAreceptorsubunitsinthemousebrainImmunostaininginmousesagittalbrainsectionsGlutamateReceptorsKainateandAMPAreceptorsareverysimilarEPSPsaregeneratedbyNa+influx海人藻酸SynapticresponsestoglutamateusuallyhavebothAMPA-andNMDA-receptormediatedcomponents

N-methyl-D-aspartate(NMDA)receptor1.

WhyfocusingontheNMDA-receptor? -Unique,multiplefeatures; -Importantphysiologicalroles; -NMDA-receptordysfunctioncanleadtobraincelldeath.2.

OverviewoftheNMDA-receptormolecularbiologyNMDAreceptorbindingsites4outsidecellGlutamateGlycineObligatoryco-agonistZinc(inverseagonist)Polyamine(indirectagonist)2insidecellMagnesium(inverseagonist)PCP(inverseagonist)

苯環(huán)己哌啶-UniquefeaturesAsallligand-operatedionchannels,theNMDA-receptorcomplexhasanagonistbindingsite.OUTINGateAgonistbindingsiteGlutamate,NMDA(agonist)But,occupationoftheagonistsitebyNMDAorglutamateisNOTsufficientto‘openthegate’.Ionophore(channel)‘Gate-opening’,requiresasecondaminoacid,glycine,tobepresentandtobindtoanallostericsiteonthereceptorcomplex.OUTINAllostericsiteforglycineGlutamateNMDA‘Gate-opening’,requiresasecondaminoacid,glycine,tobepresentandtobindtoanallostericsiteonthereceptorcomplex.OUTINGlycineCo-agonist(activator)GlutamateNMDAAllostericsiteforglycine

Glycinepotentiatesthebindingofglutamatetoitsreceptor.AllostericmodulationofNMDAreceptorsbyD-serineOUTINGlycineCo-agonist(activator)GlutamateNMDAIncontrasttootherligand-operatedionchannels,‘Gate-opening’aloneisNOTsufficientfortheNMDA-receptorcomplextoletionsthrough.Wait!Youstillcan’tgetthrough.Ca2+Na+Ca2+Why?OUTINGlycineCo-agonist(activator)GlutamateNMDAWait!Youstillcan’tgetthrough.Because,aslongasthemembraneremainspolarised,theporeofthechannelisblockedbyphysiological,extracellularconcentrationofMg2+.Mg2+Mg2+Mg2+Mg2+bindingsiteCa2+Na+Ca2+OUTINGlycineCo-agonist(activator)AllostericsiteGlutamateNMDACa2+Na+Ca2+ThemembranemustbeindependentlydepolarisedtorelievetheMg2+-blockandallowionstoflowthrough.Mg2+IndependentmembranedepolarisationOUTINGlycineCo-agonist(activator)AllostericsiteGlutamateNMDAMg2+MultipleregulatorysitesPolyaminesVoltage-dependentMg2+-blockEndogenouspolyamines(e.g.spermineandspermidine)modulatetheNMDA-receptorcomplex,withlowμMandhighμMpotentiatingandinhibitingitsfunction,respectively.**精胺亞精胺OUTINGlycineCo-agonist(activator)AllostericsiteGlutamateNMDAMg2+PolyamineMultipleregulatorysitesVoltage-dependentMg2+-blockActivationofintracellularproteinkinaseC(PKC)enhancesNMDA-receptormediatedresponses.*Phosphorylationsite*GlutamateNMDAMultipleregulatorysitesOUTINGlycineCo-agonist(activator)AllostericsiteMg2+PolyamineVoltage-dependentMg2+-blockPhosphorylationsiteRedoxsite-S=S-*NMDA-receptormediatedresponsescanbemodulatedbyredoxchanges,possiblybyoxidationandreductionofthiolgroup(s)locatedwithintheNMDA-receptorcomplex.Exposuretoreducingagents(e.g.dithiothreitol二硫蘇糖醇)potentiates,whereasoxidationreducesNMDA-receptoractivation.*NR1NR1NR2NR2?NMDAreceptorsarelargehetero-oligomericcomplexesincludingatleasttwocopiesofanNR1subunitandtwocopiesofanNR2subunit.NR2NR1GlyGluCaNa2++NMDAreceptorsubunits

NR1ubiquitous

8spliceforms

NR2Aforebrain/cerebellumNR2Bforebrain/spinalcordNR2CcerebellumNR2DThalamus/spinalcord

NR3ArareinadultNR3BspinalcordAgonistSensitivityandDeactivationRatesAmongNMDAReceptorSubtypesAgonistPotencyNR1/2ANR1/2BNR1/2CNR1/2DGlu1.70.80.70.4Glycine2.10.30.20.1Off-time,ms503002751700Immatureneurons-higheragonistsensitivity,sloweroff-rate(NR1/2B,NR1/2D)Matureneurons-loweragonistsensitivity,fasteroff-rate(NR1/2A)VOLUME15|NUMBER10|2012TheNMDAGlutamateReceptorRequiresbothNTanddepolarizationtoopenBlockedatRMPbyMg2+

DepolarizationdisplacesMg2+ions“LigandandVoltage-dependent”ChannelispermeabletoNa+,K+

andCa2+AMPAreceptorsNMDAreceptorsMg2+ion++++++Ca2+NMDAandAMPAReceptorsareColocalizedatExcitatorySynapsesBut,thenumberofAMPAreceptorsistightlyregulatedbyactivity,soamplitudeoffastcomponentoftheEPSP/Cscanberapidlyaltered!FastAMPAcomponentofEPSPSlowNMDAcomponentofEPSP1mV10secMetabotropicglutamatereceptorsmGluRsfallintooneofthreecategoriesbasedonsequencehomology,effectormechanismsand,tosomeextent,theirpharmacolgy.GroupI GroupIIGroupIIImGluR-1 mGluR-2 mGluR-4mGluR-5 mGluR-3 mGluR-6 mGluR-7 mGluR-8PLC ACACBraindistributionofmGluRreceptorsMetabotropicglutamtereceptors(Eightdifferentsubtypes)Whenlocalizedtothepresynaptictermianl,inhibitneurotransmtterreleaseWhenlocalizedtothepostsynapticmembrane,exertcomplexmodulatoryeffectsthroughspecificsignaltransductioncascades.Glutamateuptake1.

Whyfocusingonglutamateuptake?

2.

OverviewoftheglutamatetransportermolecularbiologyanddistributionBecausedeficiency(orreversal)ofglutamateisoftenproposedastheunderlyingcauseoftheexcitotoxicitythat(maybe)associatedwithneurologicaldisorders.3.

Glutamateuptake,machineryandfunctionNeuronal(Retinal)Neuronal(cerebellum)NeuronalGlialGlialCellularDistributionEAAT5EAAT4EAAC1EAAT3GLT-1EAAT2GLASTEAAT1AlternativeNamesNameExcitatoryAminoAcidTransportersPrimaryfunction:

Presynapticand/orglialneurotransmittertransporterscontrolsynapticlevelsofneurotransmitterNTGlialcellPresynapticterminalShapingsynapticresponses

pHregulation

ReversetransportBut,atsynapticlevel,glutamatetransportersmayhaveothersecondary(butpotentiallyimportant)roles.EAAT4andEAAT5mediatepresynapticinhibitionincerebellumandretinaPre-synapticNeuroneGluGluhyperpolarisationCl-Cl-FUNCTINALROLESEPILEPSYPAINDEVELOPMENTMEMORYEXCITOTOXICITY目的基因功能獲得功能缺失

小鼠遺傳操作技術(shù)原理AMPAReceptorFunctionGardenvarietyexcitatorysignalsSensoryprocessingLearningPainregulationInductionofseizuresGlutamateReceptorsNatureneuroscience5(9)2002.SpatialmemorydissociationsInmicelackingGluR1

KainateReceptorFunctionNotmuchknown,BUT...PresynapticreceptorsregulatethereleaseofNT,i.e.,Glu,GABAGlutamateReceptorsNMDAReceptorFunctionDevelopmentalPlasticity/LearningIncreasedsynapticdensityincreaseddendriticbranchingincreaseddensityofspinesincreasedneuritesproutingLearning:Long-termDepression(LTD)

Long-termPotentiation(LTP)GlutamateReceptors

NMDAReceptorFunctionPainsystem:“Wind-up”Seizures:developmentandpotentiationNeurotoxicityandapoptosisSchizophrenia?(blockade)GlutamateReceptorsFunctionalConsequencesofGlutamateReceptor/TransporterKnockoutsNMDA-NR1,NR2B-earlypost-nataldeath(1day)-respiratoryfailure,feedingimpairment,reducedLTPinhippocampalspecificNR1knockout-NR2A,C,D-normaldevelopment,reducedLTP,impairedmotorcoordinationNon-NMDA-GluR1-impairedLTP-GluR2-reducedexploratorybehavior,normalorenhancedLTP-GluR2-noQ/Rediting-severeseizures,deathatPN20Transporters-GLT1-slowergrowth,spontaneousseizures,prematuredeath-GLAST-increaseddamagefollowingischemia-EAAC-enhancedbehavioralsensitivitytoconvulsantsDevelopmentGlutamtereceptorsplayacriticalroleinneuronalPlasticityandactivity-mediatedgrowthduringbraindevelopment.NMDAReceptorsActivatedbySubventricularZoneAstrocyticGlutamateAreCriticalforNeuroblastSurvivalPriortoEnteringaSynapticNetworkNeuron65,859–872,March,2010LongTermPotentiation

lasting,activity-dependentincreaseinsynapticefficacyTime(min)-100102030EPSP(%change)0153045607590LTPinductionLTPmaintenance

NMDA-receptorantagonistsinhibittheinductionofLTPStepsinvolvedinCA1LTPNMDAreceptoractivationmustoccurInfluxofCalciumintospinesthruNMDAreceptors(andsomevoltagegatedCa＋＋channels)ActivationofCaMKinaseIIiscriticalAutophosphorylationoccursPhosphorylationofAMPAreceptors(?)InsertionofnewAMPAreceptorsintosynapseMaintenanceofLTP

(forhours,days,months)RequiresGenetranscription(CREB)ProteinsynthesisMorphologicalchanges(maybe)EnlargementofdendriticspinesSplittingofspinesfromonetotwo/lab/projects.htmOtherformsoflongtermplasticity

Longtermdepression(LTD)HippocampusStriatumCerebellumLTDinducedwith1HzstimulationatCA3-CA1synapseTheJournalofNeuroscience,July1993,13(7):2910-2918NMDAreceptoractivationisrequiredforLTDExcitotoxicity2.

WHY,HOWexcessiveglutamatergicactivationishazardoustoneurons?Definition:Neuronalexcitotoxicity:deathofneuronesarisingfromprolongedexposuretoglutamateandtheassociatedexcessiveinfluxofionsandwaterintothecell.3.

Whichabnormalitiesoftheglutamatergicsynapsemightleadtoexcitotoxicstress/injury?ATPNa+K+Na+Ca2+VoltagegatingCa2+Na+Na+AMPA/Kainate-RNMDA-RCa2+homeostasisE3Na+Ca2+EXCESSIVEFUNCTIONING[Ca2+]i+DepolarisationWecan’tcopeanymore!2.WHY,HOWexcessiveglutamatergicactivationishazardoustoneurons?IntracellularCa2+-overload[Ca2+]iCa2+-activatedProteases(cytoskeletonbreakdown)Phospholipases(releaseofarachidonicacid)NOsynthasesEndonucleases(DNAbreakdown)FreeradicalproductionDeathGLUNa+K+Ca2+VSCCDockingFusionVesicle-+-+Presynapticabnormalitiesleadingtoexcessiveexcitation

Where?When?132

Deficientglutamateuptake

OutInATPNa+K+

K+Na+

K+CGlu2Na+DeficientglutamateuptakeDeficientinactivationofglutamatergictransmissionExcitotoxicityDeficientenergysupply(ischaemia,mitochondrialdamage)LossoftheNa/K-transmembranegradient(drivingforceofthecarrier)AMPA-RPostsynapticabnormalitiesleadingtoexcessiveexcitationIonotropicreceptors(GluR)Metabotropicreceptors(mGluR)Ca2+NMDA-RNa+Na+Kainate-RGCellularmembraneGlycineNMDAGlutamateNa+Ca2+OUTINMgRedoxsitePolyaminesPhosphorylationsiteIncreasedaffinityoftheglutamatebindingsitePresynapticPostsynapticKainatereceptorsGMetabotropicGIncreaseddensityofglutamatereceptorsIncreasedexpressionofkainatereceptorsinmiceinducedpronouncedseizureactivity.PresynapticPostsynapticGLUNMDA-RAMPA-RKainate-RGGAlterationoftheionicselectivityofglutamate-operatedionophoresCa2+Na+Na+Ca2+Ca2+Ithasbeendemonstratedthat,incertainconditions,AMPA-R(normallyNa+-permeable)canbecomepermeabletoCa2+.PresynapticPostsynapticGliaNMDAAMPAKainateIonotropicreceptorsMetabotropicGGAccumulationofotherendogenousGLU-Ragonists

e.g.Quinolinate對(duì)苯二酚NMDA-receptormediatedexcitotoxicityControlExposuretoNMDA(300mM)for24hoursNeuronesincellculturearekilledbywhenNMDAreceptorsareoveractivatedFundamentalNeuroscience1999byM.J.Zigmond,F.E.Bloom,S.C.Landis,J.L.Roberts&L.R.Squire.AcademicPress,SanDiegoCA,USA.ISBN:0-12-780870-1SummarySummaryGlutmateisthemajorexcitatoryneurotransmitterinthebrain.Glutamatereceptorscomprisetwolargefamilies,ligand-gatedionchannelscalledionotropicreceptorsandGprotein-coupledreceptorscalledmetabotropicreceptors.Ionotropicglutamatereceptorsaredividedintothreeclasses,AMPAreceptors,kainatereceptors,andNMDAreceptors,whicharenamedaftersyntheticligandsthatactivatethem.TheNMDAreceptorhastwoimportantbiophysicalproperties.Becauseitishighlypermaeabletocalciumandisvoltagedependent,itonlyallowscalciumentryifthecelldepolarized.AMPAreceptorsmediatethevastmajorityofexcitatorysynaptictransmissioninthebrain,whereasNMDAreceptorsplayanimportantroleintriggeringsynapticplasticityand,whenoveractivated,intriggeringexcitotoxicity.謝謝！LifeCycleofneurotransmittersBiosynthesis&StorageReleaseReceptorActionInactivation2.BiologicalfunctionsPhysiologyPathologyAminoAcids2011SmalltransmembraneAMPAreceptorregulatoryproteins(TARPs)RoleofTARPsinAMPARERExportandSynapticTraffickingAmodelforpossibleroleofTARPsonAMPARchannelopeningNMDAReceptorsareBothSynapticandExtrasynapticandAreCoupledtoDistinctSignalingPathwaysActivityRegulatestheDistributionofNMDAReceptorsToomuchortoolittleNMDAactivitycanbetoxictoneuronsGlutamateTransporters(EAAT1-5)multiplesequencealignmentrEAAT1.-----MTKSNGEEPRMGSRMERFQQGVRKRTLLAKKKVQNITKEDVKSYLFRNAFVLLTV

rEAAT4.MSSHGNSLFLRESGAGGGCLQGLQDSLQQRALRTRLRLQTMTREHVRRFLRRNAFILLTV

hEAAT5.------------------------------------MVPHTILARGRDVCRRNGLLILSV

rEAAT2.---------MASTEGANNMPKQVEVRMHDSHLSSEEPKHRNLGMRMCDKLGKNLLLSLTV

rEAAT3.-----------------------------------MGKPTSSGCDWRRFLRNHWLLLSTV70rEAAT1.SAVIVGTILGFALR-PYKMSYREVKYFSFPGELLMRMLQMLVLPLIISSLVTGMAALDSK

rEAAT4.SAVIIGVSLAFALR-PYQLTYRQIKYFSFPGELLMRMLQMLVLPLIVSSRVTGMASLDNK

hEAAT5.LSVIVGCLLGFFLR-TRRLSPQEISYFQFPGELLMRMLKMMILPLVFSSLMSGLASLDAK

rEAAT2.FGVILGAVCGGLLRLAAPIHPDVVMLIAFPGDILMRMLKMLILPLIISSLITGLSGLDAK

rEAAT3.AAVVLGIVVGVLVRGHSELSNLDKFYFAFPGEILMRMLKLVILPLIISSMITGVAALDSN

130rEAAT1.ASGKMGMRAVVYYMTTTIIAVVIGIIIVIIIHPGKGT-KENMYREGKIVQVTAADAFLDL

rEAAT4.ATGRMGMRAAVYYMVTTVIAVFIGILMVTIIHPGKGS-KEGLH