Ca_2_Entry Through TRPC ease from RaT

GLIACa2+EntryThroughTRPC1ChannelsContributestoIntracellularCa2+DynamicsandConsequentGlutamateReleasefromRatAstrocytes3ERIKB.MALARKEY1,YINGCHUNNI2,ANDVLADIMIRPARPURA13GLIA2008,56:821-835.1.DepartmentofNeurobiology,CenterforGlialBiologyinMedicine,AtomicForceMicroscopyandNanotechnologyLaboratories,CivitanInternationalResearchCenter,EvelynF.McKnightBrainInstitu2te,UniversityofAlabama,Birmingham,Alabama;2.NationalInstituteofChildHealthandHumanDevelopment,NationalInstitutesofHealth,Bethesda,MarylandAbstractAstrocytescanrespondtoavarietyofstimulibyelevatingtheircytoplasmicCa2+concentrationandcaninturnreleaseglutamatetosignaladjacentneurons.ThemajorityofthisCa2+isderivedfrominternalstoreswhileaportionalsocomesfromoutsideofthecell.AstrocytesuseCa2+entrythroughstore2operatedCachannelstorefilltheirinternalstores.Therefore,weinvestigatedwhatrolethisstore2operatedCa2+entryplaysinastrocyticCa2+responsesandsubsequentglutamaterelease.Astrocytesexpresscanonicaltransientreceptorpotential(TRPC)channelsthathavebeenimplicatedinmediatingstore2operatedCa2+entry.Here,weshowthatastrocytesincultureandfreshlyisolatedastrocytesfromvisualcortexexpressTRPC1,TRPC4,andTRPC5.Indirectimmunocyto2chemistryrevealsthattheseproteinsarepresentthroughoutthecell;thepredominantexpressionoffunctionallytest2edTRPC1,however,isontheplasmamembrane.LabelinginfreshlyisolatedastrocytesrevealschangesinTRPCexpressionthroughoutdevelopment.UsinganantibodyagainstTRPC1wewereabletoblockthefunctionofTR2PC1channelsanddeterminetheirinvolvementinmechanicallyandagonist2evokedCa2+entryinculturedastrocytes.BlockingTRPC1wasalsofoundtoreducemechanicallyinducedCa2+2dependentglutamaterelease.Thesedataindi2catethatCa2+entrythroughTRPC1channelscontributestoCa2+signalinginastrocytesandtheconsequentgluta2matereleasefromthesecells.Z2008Wiley2Liss,Inc.Keywordsstore2operatedCa2+entry;Ca2+2dependentglutamaterelease;exocytosis;signalingClassificationnumbersofChineselibraryR741;R681.55DocumentcodeAArticleID10012117X(2008)0320176216,Ca2+,Ca2+,Ca2+Ca2+(SOC),Ca2+Ca2+(SOC)TRPC(Ca2+)Ca2+TRPC1,TRPC4,TRPC5,TRPC1,TRPCTRPC1,TRPC1TRPC1,TRPC1Ca2+;Ca2+;;671NeuralInjuryandFunctionalReconstruction,May2008,Vol.3,No.31INTRODUCTIONAstrocyteshavetheabilitytoreleaseglutamateinaCa2+2dependentmannerandsignaltoadjacentneurons(Parpuraetal.,1994).Thisglutamate2mediatedastrocyte2neuronsignalingincludesmod2ulationofsynaptictransmission[reviewedin(Araqueetal.,1999)].TheintracellularCa2+el2evationsinastrocytesaresufficientandnecessarytocauseglutamatereleaseandtheunderlyingmechanismofthisreleaseisexocytosis[reviewedin(Montanaetal.,2006)].TheCa2+forthisglu2tamatereleasecomesfromtwosources:predomi2natelyfrominternalstores,andalsofromentryofextracellularCa2+.EvidenceforthiswasindicatedbythereductionofCa2+2dependentglutamatere2leasefromastrocytesinthepresenceofthapsigar2gin,ablockerofstorespecificCa2+2ATPase,andCd2+,ablockerofCa2+entryfromtheextracellu2larspace(Huaetal.,2004).Ca2+entryacrosstheastrocyticplasmamembraneinvolvesavarietyofchannels[reviewedin(Verkhratskyetal.,1998)].Someofthese,socalled,store2operatedCachannelsareactivatedbythedepletionofinter2nalCa2+stores.Transientreceptorpotential(TRP)genes,whichhavebeenimplicatedinstore2operatedCa2+(SOC)entry,havebeenfoundinas2trocytesindicatingthatSOC/TRPactivityplaysaroleintheregulationofCa2+homeostasis(Golovi2na2005;Grimaldietal.,2003;Pizzoetal.,2001).Hence,antisenseknockdownofthecanon2icalTRP1(TRPC1)gene,significantlyreducedSOCentryinculturedastrocytesfrommouseem2bryos(Golovina2005).AlthoughhighresolutionmicroscopyunveilsthelocationofTRPC1proteinincloseproximitytotheCa2+2ATPasethatrefillsendoplasmicreticulumCa2+stores(Golovina,2005),whetherthisproteinfunctionallycontrib2utestoCa2+2dependentglutamatereleasefromas2trocytesisnotyetknown.Inthisstudy,consistentwiththepreviousfindingofTRPC1andTRPC4inembryonicculturesofmouseastrocytes(Golovina,2005),wedetectedthepresenceofTRPC1inculturedastrocytesfromneonatalratvis2ualcortices,alongwithtwootherisoformswithwhichTRPC1isknowntoformheteromultimericchannels,TRPC4andTRPC5(Hofmannetal.,2002;Strubingetal.,2001;Strubingetal.,2003);TRPC1doesnotappeartocommonlyformhomomericchannels(Strub2ingetal.,2003).Additionally,wefindexpressionoftheseTRPCsnotonlyinculturedastrocytes,butalsoinfreshlyisolatedastrocytesfromratvisualcortices.Usingratsatvariousages,wedeterminedthedevelop2mentalexpressionprofileoftheseproteinsinfreshlyi2solatedastrocytes.Lookingatthefunctionoftheseproteins,wemeasuredtheextentofSOCentryincul2turedastrocytesandfoundthatTRPC1mediatessuchanentry.Additionally,byusinganantibodydirectedatanepitopeintheporeformingregionoftheTRPC1protein,weobservedthatblockingTRPC12mediatedSOCentryresultedinreductionofagonistandme2chanicallyinducedcytoplasmicCa2+elevationsinastro2cytes.ThisalterationinCa2+responsehadconse2quencesonglutamaterelease,hamperingmechanicallyinducedreleasefromastrocytes.Becauseacuteimmu2nologicalinhibitionofTRPC1geatlyreducedCa2+2de2pendentglutamatereleasefromastrocytes,thisproteincanplayafunctionalroleinastrocyticg