医学神经生物学：Inhibitory Amino Acids

InhibitoryAminoAcids

History

GABA(Gamma-AminobutyricAcid)isanaminoacidthatwasfirstdiscoveredin1883inBerlin.In1950,RobertsandAwaparaindependentlydiscoveredthat

therewereprodigiousamountsofGABAinthemammalian

centralnervoussystem—1mgpergram.

In1957,theyfurtherdemonstratedthatchemicallysynthesized

GABAcouldinhibitthecrayfishstretchreceptor(小龙虾牵张感受器).

-AminobutyricAcid(GABA)History

ThisobservationpromptedErnstFloreytopropose

thatGABAwasactingasaninhibitoryneurotransmitterinthebrain.In1976,HedgecockfoundlysatesfromC.

elegans

exhibitedenzymaticactivityfortheGABAbiosyntheticenzyme,glutamicaciddecarboxylase(GAD).In1981,GABAAandGABABreceptorspharmacologicallydistinguished.In1987,GABAAreceptorswerecloned.In1990,GABAtransporter1wascloned.In1997,GABABreceptorswerecloned.Fluorescentmicrographofanadulthermaphrodite(雌雄同体)

stainedwithanantiGABAantiserum.Thereare26neuronsthatstainforGABA.Anterioristotherightandtherightsideofthebodyisshown.Scalebar=0.10mm.Schematicdrawingofthepositionsofthe26GABA-containingneurons.

LifeCycleofGABABiosynthesis&StorageReleaseReceptorActionInactivation2.BiologicalfunctionsofGABAPhysiologyPathologyGABA(GammaAminobutyricAcid)PrincipalInhibitoryNTBiosynthesis:Removedbyreuptake3receptortypesGABAA

(ionotropic)GABAB

(metabotropic)GABAC

(ionotropic)GluGABAGlutamicAcidDecarboxylase(GAD)andB62SynthesisandMetabolismofGABA琥珀酸延胡索酸苹果酸草酰乙酸异柠檬酸柠檬酸琥珀酸半醛a-酮戊二酸丙酮酸葡萄糖RegulationofGADapoGAD:inactiveenzyme脱辅基酶holoenzyme:activeenzymewithPLP全酶GAD67:solublecytosolicproteinsaturatedwithPLP

(吡哆醛，B6)GAD65:bothinthecytosolandasmembrane-boundformmajorityoftheapoenzymeSpeciesGeneProteinHumanGAD1(Chr2)GAD67594aaGAD2(Chr10)GAD65585aaMouseGad1(Chr2)GAD67593aaGad2(Chr2)GAD65585aaInhibitorsofGAD:Hydrazides肼类:interactionwiththeco-factorPLPAllylglycine烯丙基甘氨酸,3-mercaptopropionicacid3-巯基丙酸

:competitiveinhibitorsGAD67Caudatenucleus：尾状核;Corpuscallosum：胼胝体DeficiencyinGAD67:deathsoonafterbirth.GAD65Caudatenucleus：尾状核;DeficiencyinGAD65:spontaneousseizures.

Atransgenicmousethatexpressesthegreenfluorescentprotein(GFP)inGADpositiveneurons.bis-benzimide苯甲亚胺StorageofGABAVGAT/VIAAT:520aa,10transmembranedomains;ConferringvesicularGABAandglycinetransport;

Chaudhryetal.2002JCB.VesicularaccumulationofaminoacidsresultsfrombothagradientofmembranepotentialandpH.GABAReuptakeGATsBelongtoaFamilyofNa+/Cl--dependentNeurotransmitterTransporters(SLC6family)PlasmaMembraneGABATransportersGlycineTransportersDAT,NET,SERTOrphanTransporters

GABAReuptakeintheCNSGAT1:mainlyintheneurons,someinglialcells.GAT-2:inthebrainandkidney,transportingtheosmolyte(渗透物)

betaineaswellasGABA.

GAT3:principallyinglia,alsoinsomeneurons.GAT4:incellsofependymaandarachnoidmembrane(室管膜和

蛛网膜)inthebrainandinliver.FrontiersinCellularNeuroscienceJune2014|Volume8Thecellularandsub-cellulardistributionofGABAtransportersDistributionofGAT-1(A),GAT-2(B),GAT-3(C),VGAT(D),GABA(E)andthionine(亚氨嗪)-stainedsection(F)inthreeadjacentsectionsofdevelopingSIcortex(感觉皮层)(P5rat).Bar:100mforA–E.ABDistributionofGAT-1(A),GAT-2(B),GAT-3(C),GAD67(E),andthethionine-stainedsection(D)inthreeadjacentsectionsofSIcortexinanadultrat.cRNA;mRNATranslationModificationSortingFunctionanalysisThemodeofactionandfunctionaleffectofGABAtransportersonsynaptictransmissionFrontiersinCellularNeuroscienceJune2014|Volume8Reuptake(FunctionsofGATs)AfractionofGATsisstrategicallyplacedtomediateGABAuptakeatfastinhibitorysynapses,terminatingGABA'sactionandshapinginhibitorypostsynapticresponses;GATsmaycontributetothedysregulationofneuronalexcitabilitythataccompaniesmajorhumandiseases:epilepsy,ischemia,etc.Tiagabine/Gabitril:硫加宾Obviousphenotype:appearanceofspontaneousstrongtremorinGAT1-/-mice

A.GAT1(-/-)miceshowstrongspontaneoustremor,ataxia,andnervousnessGABAReceptorsintheCNSBenzodiazepinesensitive

GABAA GABAB GABACNeuronalexcitability(seizures)Memory ??Rapidchangesinmood(anxiety)Mood(depression)Sleep Analgesia

痛觉丧失GABAAreceptorsAnionpermeableionchannel(Cl-)Multi-SubunitComplex(pentameric)MajorinhibitorytransmitterinadultbrainTargetformanydrugsandcompoundsGABAABindingSitesGABAMuscimol(蝇蕈醇directagonist);bicuculine(荷包牡丹碱

directantagonist)Benzodiazepine(indirectagonist)Naturalinverseagonistbindshere(fear,tension,anxiety)Tranquilizingdrugs(anxiolytics):valium,librium

(安定)

(利眠宁)LikelysiteforalcoholBarbiturate(indirectagonist)Phenobarbital;pentobarbitalSteroid(indirectagonist)Picrotoxin(Antagonist):印防己毒素causesconvulsions惊厥GABAAreceptorstructurenaturereviews|neuroscience2008Table1.GABAAreceptorsubtypesa

SubunitsLocalizationPharmacologyα1β2γ2Majorsubtype(60%):synapticandextrasynapticBenzodiazepine-sensitive.Mediatessedativeandanticonvulsantactivityα2β3γ2Minorsubtype(15–20%):synapticBenzodiazepine-sensitive.Mediatesanxiolyticactivityα3βnγ2Minorsubtype(10–15%)Benzodiazepine-sensitive.Pharmacologyyetunclearα5β1,3γ2Lessthan5%ofreceptors:extrasynaptic(cerebralcortex,hippocampus,olfactorybulb)Benzodiazepine-sensitive.Mediatesmodulationoftemporalandspatialmemoryα4βnδLessthan5%ofreceptors:extrasynapticInsensitivetobenzodiazepines.Sensitivetolowconcentrationofethanolα4βnγLessthan5%ofreceptors:extrasynapticInsensitivetobenzodiazepinesα6βnδSmallpopulation:extrasynaptic(onlyincerebellum)Insensitivetobenzodiazepines.Sensitivetolowconcentrationofethanolα6β2,3γ2Lessthan5%ofreceptorssynaptic(onlyincerebellum)Insensitivetobenzodiazepinesa

Thetermbenzodiazepinereferstodiazepamandstructurallyrelatedagentsinclinicaluse.DifferentialdistributionofGABAARαsubunitvariantsintheadultmouseforebrainSubcellularDistributionofGABAAReceptorsDoublelabelcellfordifferentGABAAsubunits

2/3-10nmgoldparticles-synapticlocalization-

-20nmgoldparticles-extrasynapticlocalization-mousecerebellumModesofGABAAReceptorActivationmIPSCeIPSCSynapticExtrasynapticTonicPhasicSynapticGABAReceptors(EC5010-20

M)

(1,2,3,6)

(2,3)

2SubunitCompositionofSynapticandExtrasynapticGABAAReceptorsExtrasynapticGABAReceptors(EC50~0.5

M)Cerebellargranuleneurons

6

(2,3)

Thalamus,dentategyrus

Hippocampalpyramidal

5

3

2ExcitatoryEffectsofGABAinImmatureNeuronsHippocampalslices/pyramidalneuronsStimuluselicits4post-synapticresponsesTwooftheseareblockedbyAMPAandNMDAantagonistsTheremainingtwoareblockedbyGABAAantagonistbicucullineSoGABAAreceptorscanexciteneuronsalso-how?NKCC1Na+K+2Cl-CurrentOpinioninPharmacology2015,20:102–108GABAReceptorsGABABReceptor

MetabotropicreceptorCouplestoCa++andK+channelsviaGproteinsProduceshyperpolarizationandinhibitionofneurotransmitterreleaseResultsinslowerregulationinsensitivityofneuronalfiringMaybeimportantinmemory,mood(depression),painGABABReceptorsFirstsuspectedin1981;l-baclofen(β-对氯苯基-γ-氨基丁酸[肌松药])inducedresponsesnotblockedbybicucculine,theGABAAantagonistExpressioncloninginoocytesnotsuccessful

cDNAisolatedin1997Neuropharmacology60(2011)GABABActionsGABABGK+PostsynapticPresynapticGABABGCa+GABAGABAStructureandeffectormechanismofGABABreceptorPharmacology,BiochemistryandBehavior110(2013)174–184FrontiersinPharmacology2014SchematicpresentationofGABABreceptorinteractingproteinsandcrosstalkofGABABreceptorwithotherreceptorsBiochemicalPharmacology86(2013)GABABReceptorsandNeuropathicPainGABACReceptorLigand-gatedCl-channel(similartoGABA-A)Notatargetofbenzodiazepines,barbiturates,ethanolPhysiologicrolelessclear(atpresent)PharmacologyandStructureofGABAcReceptorsCis-4-transaminocrotonicacid:反-丁烯酸TPMPA：1,2,5,6-四氢吡啶-4-基-甲基次膦酸IS:内段OPL:外网层GABAAreceptorsarethetargetsoftherapeuticdrugsAnticonvulsantdrugs GABAAmodulatorsAntianxietyagents GABAAmodulatorsEthanolinteractionswithGABAAreceptors(post-synapticeffects)Ligand-gatedionchannels:allostericenhancement

IonchannelporeGABAbarbituratesCl-Cl-Ethanol?IntracellularExtracellularbenzodiazepinesOnepossiblemechanismforanindirectinteractionofethanolwithaligand-gatedionchannelIonchannelporeGABAEthanolIntracellularExtracellularKinasePO4ATPRo15-4513blockseffectofalcoholatGABAAreceptor昏迷癫痫GABAandPND6-77dayslaterGABAAreceptoragonistBritishJournalofPharmacology(2010)161271–287MotorControlTrendsinNeurosciences2011RewardSystemScience.2003May2;300(5620):812-5.GABAanditsagonistsimprovedvisualcorticalfunctioninsenescentmonkeysLeventhalAG,WangY,PuM,ZhouY,MaY

FormationofSerineGlucoseGlycolysis3-Phospho-glycerate3-Phospho-hydroxypyruvate3-PhosphoserineSerine(Ser)PyruvateDehydrogenaseNAD+NADH+H+Glutamatea-KetoglutarateTransaminasePhosphatase3StepsInhibits磷酸甘油酸羟基丙酮酸ConversionofSerinetoGlycineFolateTetrahydrofolate(FH4)DihydrofolatereductaseN5,N10-MethyleneFH4SerineGlycineSerinehydroxymethyltransferase(PLP-dep.)KeyintermediateinbiosynthesisofpurinesandformationofthymineImportantinbiosynthesisofhemeandporphyrinsGlycineLocatedbyradioligandbindingoccursmainlyinspinalcord&brainstemOverviewonly1receptortypeionotropicopenschannelpermeabletoCl-reverses~-75mVthereforegeneratesfastIPSPinspinalcord&brainstemReleaseinhibitor:Tetanustoxin破伤风毒素Pharmacologyagonists=glycine&alanineandtaurineantagonist=strychnine,picrotoxin(Cl-channelblocker)MolecularbiologyofglycinereceptornicotiniclikeformpentamersPhysiological/pathologicalrolesRenshawcells(脊髓前柱内侧短轴突神经元)areglycinergicspasticpatientsshowlossofstrychninebindinggeneticallyspastic(痉挛的)mousehasfaultongeneencodingtheglycinereceptordiseaseistreatedwithbaclofen(氯苯氨丁酸)PharmacologyandStructureofGlycineReceptorsGlycinereceptorssubunit:a1-4

b1-4GlycineExcitationglycineglycineGlycinereceptorGlycinereceptorHypotheticaltopologyofGLYT1GlycinereuptakeGLYT1:threeisoforms(neuronsandastrocytes)GLYT2:neuronsA:TopologyofGlyT1bandGlyT2a.N-terminusofGlyT2a,over200amino-acidsB:Determinationofthechargetoglycineratio.glycineuptakecurrentundervoltage-clamp

2)radio-labeledglycineuptakePlottingtheamountofchargeasafunctionofglycineuptake.Affinitiesofglycine‘receptors’andtransporters(left)andthecomparisonbetweentheseaffinitiesandthegradientsthatcanbemaintainedbyGlyT2aandGlyT1bifequilibriumisreached.*Hindfeetclasping“phenotypeinbothGlyT2(−/−)miceandGAT1(−/−)mice

A.GAT1(-/-)miceshowstrongspontaneoustremor,ataxia,andnervousnessB.GlyT2(-/-)miceshowstrongspontaneoustremor

(Jesusetal.Neuron,2003,40:797-806)Obviousphenotype:appearanceofspontaneousstrongtremorinGAT1-/-andGlyT2-/-mice

SummaryGABAIsthemostimportantinhibitoryneurotransmitterinthecentralnervoussystem(CNS)Presentin60-70%ofallsynapseswithinCNSBindstothreemajorreceptortypesGABAA;GABAB;GABACPlaysanimportantrolein:Sensitivityforneuronalfiring(seizuredisorders)Mood,cognition,pain,sleep,andmovementdisordersSummaryglycineGlycinemadefromglucoseviaaminoacidserine.High-affinityuptakesystemremovesglycinefromsynapse.SharesavesicularpumpwithGABA,VGATGlycineanditspumpfoundinhighlevelsinspinalcord,inneuronspresynaptictostrychnine-sensitiveglycinereceptor-chloridechannel.Summary谢谢！GAD65GAD67Highlyschemat