Proc.Natd.Acad.Sci.USAVol.89,pp.11051-11055,November1992CellBiologySti - PDF document

Proc.Natd.Acad.Sci.USAVol.89,pp.11051-11055,November1992CellBiologyStimulatoryandinhibitoryregulationofcalcium-activatedpotassiumchannelsbyguaninenucleotide-bindingproteinsHIROAKIKUME*,MICHAELP.GRAZIANOt,ANDMICHAELI.KOTLIKOFF*t*DepartmentofAnimalBiology,SchoolofVeterinaryMedicine,UniversityofPennsylvania,3800SpruceStreet,Philadelphia,PA19104-6046;andtDepartmentofMolecularPharmacologyandBiochemistry,MerckSharp&DohmeResearchLaboratories,Rahway,NJ07065CommunicatedbyAlfredG.Gilman,August27,1992ABSTRACTTheregulationofmembraneionchannelsbyguaninenucleotide-bindingproteins(Gproteins)hasbeendescribedinnumeroustissues.Thisregulationhasbeenshowntoinvolvethemembrane-delimitedstimulatoryactionofGproteinsonionchannels.Wenowshowthatsinglecalcium-activatedpotassiumchannels(Kcachannels)inairwaysmoothmusclecellsarebothstimulatedandinhibitedbyGproteinsinmembranepatches.WedemonstratethattheP-adrenergicagonistisoproterenolstimulateschannelactivityviatheasubunitofthestimulatoryGproteinofadenylylcyclase,Gs,andthatchannelopeningisinhibitedbytheactionofthemuscarinicagonistmethacholine,actingviaapertussistoxin-sensitiveGprotein.Isoproterenolstimulatedandmethacholineinhibitedchannelactivityinthesameoutside-outpatcheswhenGTPwaspresentatthecytosolicsurfaceofthepatch.Ininside-outpatches,additionofGTPandguanosine5'-[y-thioltriphosphate(GTP[yS])augmentedchannelactivitywhenisoproterenolwasincludedinthepatchpipette,andinhibitedchannelactivitywhenmethacholinewasincludedinthepipette.Consistentwiththeseresults,inthepresenceofGTP[yS],theasubunitofG.(a.sGTP[ySjcomplex)openedKcachannelsinadose-dependentmanner,whereasinthepresenceofguano-sine5'-[J3-thioldiphosphate,ashadnoeffect.Bycontrast,applicationofactivateda,ora.proteinsdidnotinhibitchannelactivityininside-outpatches,indicatingthatchannelinhibitionismorecomplexthanasimpleasubunit/channelinteraction,similartothecomplexinhibitoryregulationofadenylylcyclase.Theseresultssuggestthathormonalregula-tionofKcachannelssharessubstantialfeatureswiththeregulationofadenylylcyclaseanddemonstratethatasingleionchannelmayserveastheregulatorytargetforthemembrane-delimitedactionofstimulatoryandinhibitoryGproteins.Moreover,theydemonstrateapotentiallyimportantfunctionalpathwaybywhich3-adrenergicandotherG-linkedreceptorsstimulaterelaxationofsmoothmuscle,independentofcAMP-dependentproteinphosphorylation.Large-conductance,calcium-activatedpotassiumchannels(Kcachannels)areubiquitousmembraneionchannelsinsmoothmuscle,brain,andothertissues.OnemechanismofKcachannelregulationinvolvestheindirectactivationofthechannelbyadenylylcyclase-linkedreceptorstimulation,leadingtophosphorylationofthechannelbycAMP-dependentproteinkinase(1-4).Insmoothmuscle,activationofadenylylcyclase-linkedreceptorsresultsinamarkedaugmentationofpotassiumchannelactivity(5-7),whichisaprincipalmechanismofrelaxationofvascularandnonvas-cularsmoothmuscle(8-11).Forexample,P2-adrenergicreceptoragonistsarewidelyusedclinicallytorelaxairwaysmoothmuscle,anditisknownthatreceptorstimulationresultsinanincreaseinKCachannelactivityinairwaysmoothmusclecells(5)andthatthesechannelsplayanimportantfunctionalroleinP-adrenergicbronchodilation(12,13).Conversely,muscarinicagonists(andothercontrac-tileagents)inhibitpotassiumconductancesinsmoothmuscle(14-18),andthisfunctionalantagonismbetweenadrenergicandmuscarinichormoneactionconvergesonasinglepotas-siumcurrent,theMcurrent,innonmammaliansmoothmusclecells(8).AlthoughKc,channelactivationbycAMP-dependentproteinkinasehasbeendocumentedinsmoothmuscle,membrane-delimitedmechanismsofchannelregu-lationanalogoustothosedescribedinothercells(19-23)havenotbeenexplored.Wereportthatf-adrenergicstimulationactivatessingleKcachannelsindependentlyofchannelphos-phorylation,viathemembrane-delimitedactionoftheasubunitofthestimulatoryguaninenucleotide-bindingpro-tein,G..Moreover,wedemonstratefunctionallyantagonis-tic,hormone-linkedstimulatoryandinhibitoryregulationofKcachannelsatthesingle-channellevel,indicatingthatthechannelisregulatedbystimulatoryandinhibitoryGproteinsinamannersimilartotheregulationofadenylylcyclase(24-27).MATERIALSANDMETHODSCellDissociationandElectrophysiology.Unitarycurrentsfrommembranepatcheswererecordedbystandardmethods(28)fromporcine,canine,andferrettrachealmyocytesthathadbeendisaggregatedasdescribed(29).Cellsfromallspeciesbehavedidentically;allexperimentswereperformedatleastonceinpatchesfromeachspecies.Currentswerefilteredat1kHzanddigitizedat5kHz[-3decibels(dB)]foropen-stateprobabilityanalysis.Displayedrecordswerefil-teredat250or500Hzanddigitizedat0.5or1kHzfordatacompression.Thecytosolicsolutionusedwas126mMKCI/5mMNaCl/1mMMgCl2/2.5mMEGTA/10mMHepesadjustedtopH7.2(KOH);1mMCaCl2wasaddedtoachieveafreeCa2+concentrationof-0.1iAM(30).Someexperi-mentswereperformedwith5mMMgCl2;nodifferenceinresponsewasobserved.Theextracellularsolutionwas125mMNaCl/5mMKCl/1mMCaCl2/1mMMgCl2/10mMHepesadjustedtopH7.4(NaOH).Single-channelanalysiswasperformedusingsoftwareroutinesprovidedbyM.Nelson(UniversityofVermont).Thevoltagedependenceofopen-stateprobabilitywasanalyzedbyfittingthedatatotheBoltzmannequation:nP.=1/{1+exp[(V-Vm)/k]}.Ineachexperimentalconditionevaluatingtheeffectofaddedago-nistsorguaninenucleotides,nP.wasdeterminedovera3-to5-minperiodbeforeandimmediatelyfollowingtheadditionandAnP0(nP0experimental/nPocontrol)wasdetermined;analyzedperiodswereidenticalinanygivenexperiment.Statisticalcomparisonsweremadebyone-wayanalysisofvariance;dataareexpressedasmean±SE.Abbreviations:Kcachannel,calcium-stimulatedpotassiumchannel;ATP[B,yNH],adenosine5'-[B,y-imido]triphosphate;GTP[yS],gua-nosine5'-[ythio]triphosphate;GDP[flS],guanosine5'-[-thio]di-phosphate.Towhomreprintrequestsshouldbeaddressed.11051Thepublicationcostsofthisarticleweredefrayedinpartbypagechargepayment.Thisarticlemustthereforebeherebymarked"advertisement"inaccordancewith18U.S.C.§1734solelytoindicatethisfact. 11052CellBiology:Kumeetal.GuanineNudeotldes.Recombinanta.(as-short,the45-kDaform)wasexpressedinandpurifiedfromEscherichiacoli(31).a.(30,uM)waspreincubatedwith30uMguanine5'-[y-thio]triphosphate(GTP[yS])or5'-(.-thioldiphosphate(GDP[f3S])in50mMHepes/8mMMgCl2/1mMEDTA/1mMdithiothreitolatpH8for30minat230C.assubunitsweredilutedinthesamebufferto100timesthefinalconcentrationandfrozeninaliquots.Forpertussistoxin(P-6659;Sigma)treatment,dissociatedcellswereincubatedwithtoxin(0.1,ug/ml)for4hrat370C.Thistreatmentcompletelyblocksmuscarinicchannelinhibitioninoutside-outpatches(18).cAMP-dependentproteinkinasewasinhibitedbyadditionofA-kinaseinhibitor(P-5636;Sigma)at5,ug/ml,sufficienttoinhibitallkinaseactivityfrom-5mgoftissue(32).Theinhibitorwasaddedtothebathafterinside-outpatchforma-tion,andthepatchwasincubatedfor30min.RESULTSStimulationandInhibitionbyAgonistsinOutside-OutPatches.WehavepreviouslydemonstratedGprotein-dependentinhibitionofsingleKcachannelsininside-outandoutside-outpatches(18).ToexamineanalogousstimulatorycouplingmechanismsbetweenhormonereceptorsandKcachannels,channelactivitywasexaminedinoutside-outpatchesunderconditionsofphysiologiccalciumconcentra-tionandGTP(100,uM)atthecytosolicpatchsurface,beforeandafterexposuretothe(3-adrenergicagonistisoproterenol.Fig.1Ashowsthatbathadditionof11AMisoproterenoltooutside-outpatchesstimulatedchannelactivity;followingdrugwashout,channelactivitywasinhibitedbycharybdo-toxin(100nM),aspecificinhibitorofthelarge-conductanceKc,channelinthistissue(33).Insevensimilarexperiments,isoproterenolstimulatedchannelactivity5.1+0.5-foldandproducedanapparentleftshiftinthevoltage/nPOrelation-ship(Fig.1B),althoughthiseffectcouldnotbediscriminatedfromashiftinthemaximalopen-stateprobabilityofthechannel,sincethepatchescontainedtoomanychannelstopermitaccurateestimationofthemaximumnPO.AlthoughATPwasnotaddedtothepatchpipette,wesoughttoeliminatethepossibilityofchannelphosphorylationthroughalocalizedactivationofcAMP-dependentproteinkinasesupportedbyendogenousATP,byperformingexperimentsinthepresenceofthenonmetabolizableATPanalogueaden-osine5'-[3,y-imidoldiphosphate(ATP[B,yNH])toinhibitphosphorylation.ChannelstimulationwasobservedinthepresenceofATP[3,yNH](1mM)inthepatchpipette;intwosuchexperiments,meanstimulationwas4.5-fold.Functionallyantagonisticchannelregulationwasdirectlydemonstratedbysequentialadditionofstimulatoryandin-hibitoryhormonestothesameoutside-outpatches.Fig.2Ashowsthatadditionofisoproterenol(1,uM)stimulatedchan-nelactivity,and,followingdrugwashout,themuscarinicagonistmethacholine(10IAM)decreasedchannelactivity.Receptor-linkedinhibitoryandstimulatorymodulationofchannelactivitywasnotsequence-dependent;resultsfromanexperimentinwhichchannelactivitywasinhibitedbymethacholineandthenstimulatedbyisoproterenolareshowninFig.2B.Fig.2Csummarizesoutside-outexperimentsthatdemonstratedfunctionallyantagonistichormoneactiononchannelactivity.Inhibitionofchannelactivitybymeth-acholine(10or50,uM)wassimilartothatreported(18);channelactivityinfivepatcheswasinhibitedbymeth-acholineto0.26±0.05ofcontrolvalues,andthisinhibitionwasGprotein-dependentandpertussistoxin-sensitive.StimulationandInhibitionbyGuanineNuceotidesinInside-OutPatches.Wenextsoughttodemonstratemembrane-delimitedG-proteinactionsonKcachannelsbytheadditionofexogenousguaninenucleotidestoinside-outpatches.Consistentwithoutside-outexperiments,additionofguanineAGTP/100AMi/ControlProc.NatLAcad.Sci.USA89(1992)ISO(1tiM)~~~~~~~~~LLLLU_biCotrlh:.iml10__;-i4Wash(8min)ICHTX(100nM)iB0.81-GTPj(10oM0cl0.40.2-ISO(1AM)Control-40-2002040Holdingpotential,mVFIG.1.IsoproterenolstimulatesKcachannelactivityinoutside-outpatches.(A)Additionofisoproterenol(ISO)toanoutside-outpatchopensKcachannels.Channelactivationwasreversiblefol-lowingbathwash;charybdotoxin(CHTX),aspecificinhibitorofthelarge-conductanceKc.channel,blockedchannelactivity.Traceshowscontinuousrecordingwithadditionartifactsblanked,exceptforan8-minwashasindicated.Calibration:10s,3pA.Caninecell0816C;holdingpotential,0mV.(B)Open-stateprobabilityversusholdingpotentialforpatchshowninA.Stimulatedchannelactivityappearsatallvoltages;nP.wasdeterminedover30sateachpotential.Boltzmannfitstothedata(lines)indicatetheapparentshiftinthevoltage/open-stateprobabilityrelationship.nucleotidesstimulatedchannelactivitywhenisoproterenolwaspresentattheextracellularmembranesurface.Fig.3AshowsthatadditionofGTP[yS](100AuM)toaninside-outpatch(1juMisoproterenolinthepipette)resultedinarapidstimulationofchannelactivity;thekineticsofchannelacti-vationwerequiterapid(meantimetomaximumwas2.5min).Fig.3Bsummarizesaseriesofexperimentsinwhichguaninenucleotideswereaddedtoinside-outpatchesinthepresenceorabsenceofagonists.AdditionofGTP[yS]toinside-outpatchesintheabsenceofanyreceptoragonistresultedinaslightincreaseinchannelactivity(1.23-foldincrease,10patches),consistentwithpreviousreports(34).SinceKcachannelsareinhibitedbymuscarinicreceptorstimulationviaapertussis-sensitiveGprotein(18),wereasonedthattheeffectofGTP[yS]alonewasconsistentwiththeactivationofbothstimulatoryandinhibitoryG-proteinregulatorsofKcachannels,resultinginanaggregatechannelstimulation.Ex-posureoftheexternalpatchmembranetothea-agonistresultedinmarkedaugmentationofguaninenucleotide-stimulatedchannelactivity(GTP,3.92+0.91-foldstimula-tion,4patches;GTP[yS],9.55+2.25-fold,5patches).WeI.mij-1-1-[-jk,ahi.Id..j Proc.Natl.Acad.Sci.USA89(1992)11053AGTPA\aouumvl/ISO({1AM:)Control111.-1;.14.1,.S..lea.1i|timi1i10IWashAidI1:11IIIJfillAi'l,IJLIIJLITlLLLB0.100.010.001C1o0.1JlmACH(10AM)GTP\(100ILM)/ISO(1AM)mACH(10/LM)**-0bb*0bb0b01020Time,mm30Time,miB0400.1-n=5n=4GTP[yS]GTPGTP[yS]GDP[BS]GTPn=5n=5TISOmACHCGTPAGDPCSmACHmACHFIG.2.StimulationandinhibitionofKc.channelactivityinthesameoutside-outpatch.(A)Exposureofanoutside-outpatchtoisoproterenol(ISO)stimulateschannelopen-stateprobability,whereas,followingwash,methacholine(mACH)inhibitsactivity.Traceshowscontinuousrecordingofagonistadditionwithadditionartifactsblanked(4minofwashrecordingwasdeletedatbeginningoflasttrace).Inthisexperiment,control,isoproterenol,control,andmethacholinenP.valueswere0.011,0.062,0.0097,and0.0006,respectively.Calibration:10s,3pA:Porcinecell0304C;holdingpotential,0mV.(B)Open-stateprobabilityversustimeforanexperimentsimilartoA,withagonistaddedinreverseorder,indicatingthatmodulationofchannelactivitydoesnotdependonsequenceofhormoneaction.ConditionsasinA.Ferretcell0228B.(C)Summaryofoutside-outexperimentsdemonstratingfunctionalantagonismofagonists,andabolitionoftheinhibitorycouplingbypretreatmentwithpertussistoxin(PTX)oradditionofGDP[PS]tothepipettesolution.Outside-outpatcheswereexposedtoasingleagonist,orbothagonistswithwashoutbetweenexposure,inwhichcaseAnP.wascalculatedrelativetothecontrolstatebeforeadditionofeachagonist.interprettheseresultstoindicatethattheinclusionofthe3-adrenergicagonistpromotesGDPreleasefromG,proteinsboundto(-adrenergicreceptors,similartoresultsobtainedusingreconstituted-3-adrenergicreceptorsandG,(35,36),FIG.3.Guaninenucleotidesstimulatechannelactivityininside-outpatchesexposedtoisoproterenol.(A)AdditionofGTP[yS](100AsM)toaninside-outpatchcontainingisoproterenolresultsinarapidincreaseinchannelopen-stateprobability.Holdingpotential,0mV;cytosolicsolutioncontained1mMMgCl2.Patch0917A.(B)Ratioofchannelactivitybeforeandafterguaninenucleotideadditiontoinside-outpatches.Intheabsenceofreceptoragonist,GTP[-yS]stimulatedchannelactivityslightly.Inthepresenceofisoproterenol(ISO,1MM),stimulationbyGTP[yS]wasgreatlyenhanced.Fol-lowingpreexposureofpatchestomethacholine(mACH,50MAM),additionofGTPresultedintheoppositeeffectonchannelactivitycomparedtowhentheP-receptoragonistwaspresent(comparebars2and5),demonstratingtheinhibitorycouplingbetweenmuscarinicreceptorandKcapreviouslyreported(18).TheseresultssuggestthattheeffectofGTPIvS]onKcainuntreatedpatchesistheaggregateeffectofactivationofstimulatoryandinhibitoryGproteins.GTP[yS]andGTPwereaddedat100MAMinallconditions;GDPLBS]was1mM.andanalogoustotheeffectofguaninenucleotide-releasingproteinsinothersystems(37).Therapidactivationkineticsobservedinthepresenceoftheagonist(Fig.3A)arealsoconsistentwiththisnotion.Moreover,infurthersupportoftheinterpretationthatthereceptoragonistpromotesGDPreleasefromGproteinsboundonlytothatreceptor,preex-posureofpatchestothemuscarinicagonistmethacholine(50ALM),whichisknowntobecoupledtoGiactivityinthistissue(38),resultedinanoppositeeffectonchannelactivityuponadditionofGTP(Fig.3B;comparebars2and5).StimulationofChannelActivitybyRecombinanta.TounequivocallydemonstratetheroleofGproteinsinthemembrane-delimitedstimulationofKc,channelactivity,recombinanta,proteinswereappliedtoinside-outpatches.Fig.4Ademonstratesanexperimentinwhichas-GTP[yS]augmentedchannelactivityinadose-dependentfashion.Meanstimulationwas5.30+0.79-fold(11patches)at100pMand15.6+4.24-fold(7patches)at1nMandcausedanapparentleftwardshiftinthevoltagedependenceofchannelopen-stateprobability(Fig.4B),similartotheeffectofadditionofisoproterenoltooutside-outpatches(Fig.1B)orCellBiology:Kumeetal.041 Proc.NatLAcad.Sci.USA89(1992)B1.0IControlasI111111EMm0.80.6GTP['yS](100pM)IIJImtsL{I-asGTP[yS](1nM)14i-IControla,*GDP[13S](10nM)I1--UJLIllI1~111IIIIIII[III1,111111i3Controlas*GTP[yS](100pM)ATP[/3,yNH](1mM)aIILllkiiIa0.40.20.0-a.GTP[yS](100pM)Holdingpotential,mVCFIG.4.a,activatesKC,channelsinaphosphorylation-independentmanner.(A)(Top)Additionofa,-GTPhyS]toaninside-outpatchresultsinarapid,dose-dependentincreaseinchannelactivity.Afteradditionof1nMa,.GTPEySI,noclosedstateisobservedforlongperiods.Patch0627.(Middle)Adifferentpatchexposedtothesamea.(10nM)thathadbeenreactedwithGDP[S]indicatestheGTP-dependentactivityofthesubunit.Patch0709.(Bottom)StimulationofKc,activitywith100pMaa.GTPhyS]occursinthepresenceof1mMATP[P,yNH]usedtocompetewithendogenousATP.Patch0829.Alltracesarecontinuousrecordingsexceptforblankingofadditionartifact.Calibration:10s,5pA.Porcinecells;holdingpotential,0mV:(B)Open-stateprobabilityversusvoltagebeforeandafterexposureofaninside-outpatchto100pMa,.GTP[yS];noteshiftinvoltagedependence.SmoothlinesareBoltzmannfitstothedata.(C)Summaryofexperimentsusingasubunits.asGTPEyS]increasednP.inadose-dependentmanner.Channelstimulationof100pMasGTP*ySIwasnotsignicantlyalteredbypreexposureofthepatchtoATPi,yNHI(1mM;P�0.5)orincubationwithA-kinaseinhibitor(5,ug/mlfor30min;P�0.5).ofguaninenucleotidestoinside-outpatches.ThestimulatoryeffectcouldnotbeexplainedbyeffectsofunreactedGTP['yS]inpicomolarconcentration,sinceininside-outpatches100,uMGTP[yS]hadonlymodeststimulatoryeffects(Fig.3B)andsincestimulationwasalsoobservedafterpreincubationofthepatchwith1mMGDP(meanstimulationin4patcheswas5.70.62-foldat100pMa,-GTP[yS]).Experimentsdemonstratingtheeffectofrecombinanta.proteinsaresummarizedinFig.4C.Additionofa,-GDP[(3S]hadnostimulatoryeffect,andboilinga.-GTP[yS]eliminatedstimu-latoryactivity(5patches,datanotshown).Channelactiva-tionwasequivalentinthepresenceofATPJyNH](1mM)oraninhibitorofcAMP-dependentproteinkinase(Fig.4C).Toeliminatethepossibilitythata,effectsonKcaweremediatedbyalocalizedrisein[Ca2+]throughastimulationofcalciumchannelsinthepatch,weexaminedtheeffectofa.underconditionsinwhich[Ca2+]pjptt,,was"100timeslowerthan[Ca2+Ibath(2.5mMEGTA,0Ca2+inpipette).Infivesuchexperiments,as-{TP[ySIstimulationwasequiva-lent(14.6+2.32-foldstimulationat1nMa).Bycontrast,additionofpurifiederythrocytea1,oraproteinsthatactivatecardiacpotassiumchannelsatpicomolarconcentrations(20,39),hadnoeffectonchannelactivity(Table1).Itisunlikelythatthelackofeffectofpertussistoxin-sensitiveasubunitswasduetotissuespecificityofthesubunits,sinceahetero-geneousmixtureofbraina/a.proteinsalsowaswithouteffect(Table1).DISCUSSION(3-Adrenergicagonistsareofclinicalimportanceasatreat-mentforbronchialasthmaandotherdisorders.TheactionsoftheseagentsaregenerallybelievedtobeassociatedwiththeGprotein-dependentstimulationofadenylylcyclaseandthephosphorylationoftargetproteinsbycAMP-dependentproteinkinases.Ourresultsshowthat,B-adrenergicagonistscanstimulateKcachannelsbyamembrane-delimitedactionofa,thatdoesnotrequirecAMP-dependentphosphoryla-tion.Channelswereactivatedbyphysiologicalconcentra-tionsofisoproterenolinoutside-outpatches(Figs.1and2)andbyguaninenucleotides(Fig.3)andGTP[yS]-activatedar(Fig.4)ininside-outpatches.Themembrane-delimitedef-Table1.InhibitionofKcaisnotmediatedbyaiora0proteinsProtein(100pM)nAnPoai241.31±0.16aij381.32+0.15ac190.95±0.12ai/ao40.95±0.18Channelactivitywasnotalteredfollowingexposureofinside-outpatchestoGTPEyS]-activatedaiora.proteins.Conditionswereidenticaltothoseinexperimentsusinga,.Purifiederythrocyteai-2,aij3,anda..1proteins(40)kindlyprovidedbyJ.CodinaandL.Birnbaumer.ai/aowasahighlypurifiedmixtureofasubunitsfrombrain(41)andkindlyprovidedbyP.Casey.AControl4011054CellBiology:KumeetaL Proc.Natl.Acad.Sci.USA89(1992)11055fectsofG,demonstratedherearelikelytobephysiologicallyrelevantforseveralreasons.(i)Relativelylowconcentra-tionsofisoproterenolstimulatedchannelactivityinoutside-outpatchesindependentlyofchannelphosphorylationunderphysiologicalconditionsofcalciumatthecytosolicmem-branesurface,indicatingthatreceptor-channelcouplingislikelytooccurintheintactcell.(ii)Channelstimulationwasreadilydemonstratedininside-outpatchesbyusingexoge-nousguaninenucleotidestoactivateendogenousGproteinsinthepatch.(iii)Charybdotoxin,aspecificblockerofKcachannelsinthistissue,antagonizedtheactionoff3-agonists,indicatingthatthesechannelscompriseanimportantfunc-tionalrolein(-adrenergicrelaxationofairwaysmoothmus-cle(12,13).Thusthemembrane-delimitedG-proteinregula-tionofthesechannelsislikelytoplayanimportantroleintheactionofG,-linkedreceptorfunctioninsmoothmuscle.Ourdatadonotexcludeadditionalchannelregulatorymecha-nisms,similartotherecentlyreportedstimulatoryeffectsof(3ysubunitsonspecificadenylylcyclaseisoforms(25).Kc,channelsarealsoactivatedbycAMP-dependentpro-teinkinasephosphorylationinairwaysmoothmuscle(5).Thus,ourresultsestablishasecondexampleofanionchannelstimulatedbydualpathways-i.e.,bymembrane-delimitedG-proteinactionsandbyGprotein-dependentphosphorylation-analogoustotheregulatorycontrolpro-posedforcardiaccalciumchannels(22,42,43).Whereasthevulnerabilityofcalciumchannelstopatchexcisionmakesthestudyofthisregulationdifficult,Kcachannelsshouldprovideamorestableandconvenientexperimentalpreparationfortheexaminationofdualstimulatorypathwaysofchannelregulation.Regulationisreadilyobservedoff-cell,andchan-nelactivityisstableformanyhours.Finally,wehavedemonstratedtheconvergenceofexcit-atoryandinhibitoryG-proteineffectsonthesameionchannel(i.e.,Kcachannels)atthesingle-channellevel.Sincepatchesalwayscontainmultiplechannels,ourdatadonotdefinitelydemonstratethatthesamechannelproteinissubjecttobothstimulatoryandinhibitoryGprotein-dependentmodulation;itispossiblethatdifferentsubtypesofKcachannelswiththesamebiophysicalandpharmacologicalproperties,butdiffer-entregulatorysites,exist.G0-linkedreceptoragonistsstimu-lated,andG1-linkedreceptoragonistsinhibited,channelsinthesameoutside-outpatches(Fig.2AandB).Byexploitingtheguanine-nucleotidereleasingactivityofagonist-receptorcoupling,wewereabletodemonstratethatadditionofguaninenucleotidesproducesinhibitionorstimulationofchannelactivityininside-outpatches,dependingontheagonistthathasbeenpreexposedtotheextracellularmembranesurface.Intheabsenceofagonist,additionofGTP[yS]stimulatedchan-nelactivityslightly,consistentwithpreviousreportsofMg2+-dependentGTPEyS]stimulationofKcachannelsreincorpo-ratedinplanarlipidbilayers(34).Similarstudiesdemonstrat-ingGTP-dependentstimulationandinhibitionofadenylylcyclasehavebeenreported(44).Whereasinhibitionofchannelactivitybyapertussistoxin-sensitiveGproteincouldbedemonstratedinoutside-outandinside-outpatches,neitheranora0proteinsinhibitedchannelactivityininside-outpatches.Unlikea,,purifiedorrecombinantai/a0proteinsalsofailtoinhibitadenylylcyclase,andtheirlackofeffectonKc,activitymaysuggestanindirectinhibitionofthechannel,similartothatproposedfortheinhibitionofadenylylcyclasebyG1(24-27).Thustheregulatoryactionsofinhibitoryandstimu-latoryGproteinsonKcachannelsarereminiscentoftheirregulationofadenylylcyclaseandmayindicateadegreeofstructuralhomology(45)andcoordinatedregulationbetweenthesemembraneproteins.andM.Pringforhelpfulcomments;andA.Gerstelfortechnicalassistance.ThisworkwassupportedbyNationalInstitutesofHealthGrantsHL41084andHL45239.1.DePeyer,J.E.,Cachelin,A.B.,Levitan,I.B.&Reuter,H.(1982)Proc.Nadl.Acad.Sci.USA79,4207-4211.2.Ewald,D.A.,Williams,A.&Levitan,I.B.(1985)Nature(London)31S,503-506.3.Levitan,E.S.&Kramer,R.H.(1990)Nature(London)346,545-547.4.Chung,S.,Reinhart,P.H.,Martin,B.L.,Brautigan,D.&Levitan,I.B.(1991)Science253,560-562.5.Kume,H.,Takai,A.,Tokuno,H.&Tomita,T.(1989)Nature(London)341,152-154.6.Sadoshima,J.,Akaike,N.,Kanaide,H.&Nakamura,M.(1988)Am.J.Physiol.255,H754-H759.7.Carl,A.,Kenyon,J.L.,Uemura,D.,Fusetani,N.&Sanders,K.M.(1991)Am.J.Physiol.261,C387-C392.8.Sims,S.M.,Singer,J.J.&Walsh,J.V.,Jr.(1988)Science239,190-193.9.Standen,N.B.,Quayle,J.M.,Davies,N.W.,Brayden,J.E.,Huang,Y.&Nelson,M.T.(1989)Science245,177-180.10.Nelson,M.T.,Huang,Y.,Brayden,J.E.,Hescheler,J.&Standen,N.B.(1990)Nature(London)34,770-773.11.Daut,J.,Maier-Rudolph,W.,vonBeckerath,N.,Mehrke,G.,Gunther,K.&Goedel-Meinen,L.(1990)Science247,1341-1344.12.Jones,T.R.,Charette,L.,Garcia,M.L.&Kaczorowski,G.J.(1990)J.Pharmacol.Exp.Ther.255,697-706.13.Miura,M.,Belvisi,M.G.,Stretton,C.D.,Yacoub,M.H.&Barnes,P.J.(1992)Am.Rev.Respir.Dis.146,132-136.14.Benham,C.D.&Bolton,T.B.(1986)J.Physiol.(London)381,385-406.15.Sims,S.M.,Vivaudou,M.B.,Hillemeier,C.,Biancani,P.,Walsh,J.V.,Jr.,&Singer,J.J.(1990)Am.J.Physiol.258,G794-G802.16.Cole,W.C.,Carl,A.&Sanders,K.M.(1989)Am.J.Physiol.257,C481-C487.17.Toro,L.,Amador,M.&Stefani,E.(1990)Am.J.Physiol.256,H912-H915.18.Kume,H.&Kotlikoff,M.I.(1991)Am.J.Physiol.261,C1204-C1209.19.Yatani,A.,Codina,J.,Brown,A.M.&Birnbaumer,L.(1987)Science235,207-211.20.Codina,J.,Yatani,A.,Grenet,D.,Brown,A.M.&Birnbaumer,L.(1987)Science236,442-445.21.Neer,E.J.&Clapham,D.C.(1988)Nature(London)333,129-134.22.Yatani,A.,Codina,J.,Imoto,Y.,Reeves,J.P.,Birnbautmer,L.&Brown,A.M.(1987)Science238,1288-1292.23.Cerbai,E.,Klockner,U.&Isenberg,G.(1988)Science240,1782-1783.24.Katada,T.,Kusakabe,K.,Oinuma,M.&Ui,M.(1987)J.Biol.Chem.262,11897-11900.25.Tang,W.&Gilman,A.G.(1991)Science254,1500-1503.26.Gilman,A.G.(1987)Annu.Rev.Biochem.56,615-649.27.Codina,J.,Yatani,A.,VanDongen,A.M.J.,Padrell,E.,Carty,D.,Mattera,R.,Brown,A.M.,Iyengar,R.&Birnbaumer,L.(1990)inGProteins,eds.IyengarR.&Bimbaumer,L.(Academic,SanDiego),pp.267-294.28.Hamill,0.P.,Marty,A.,Neher,E.,Sakmann,B.&Sigworth,F.J.(1981)PflagersArch.391,85-100.29.Kotlikoff,M.I.(1988)Am.J.Physiol.254,C793-C801.30.Fabiato,A.(1988)MethodsEnzymol.157,378-417.31.Graziano,M.P.,Freissmuth,M.&Gilman,A.G.(1989)J.Biol.Chem.264,409-418.32.Gilman,A.G.(1970)Proc.Nail.Acad.Sci.USA67,305-312.33.Boyle,J.P.,Tomasic,M.&Kotlikoff,M.I.(1992)J.Physiol.(London)447,329-350.34.Toro,L.,Ramos-Franco,J.&Stefani,E.(1990)J.Gen.Physiol.96,373-394.35.Brandt,D.R.&Ross,E.M.(1986)J.Biol.Chem.261,1656-1664.36.May,D.C.&Ross,E.M.(1988)Biochemistry27,4888-4893.37.Bourne,H.R.,Sanders,D.A.&McCormick,F.(1991)Nature(London)349,117-127.38.Sankary,R.M.,Jones,C.A.,Madison,J.M.&Brown,J.K.(1988)Am.Rev.Respir.Dis.138,145-150.39.Yatani,A.,Mattera,R.,Codina,J.,Graf,R.,Okabe,K.,Padrell,E.,Iyengar,R.,Brown,A.M.&Birnbaumer,L.(1988)Nature(London)336,680-682.40.Codina,J.,Hildebrandt,J.D.,Sekura,R.D.,Birnbaumer,M.,Bryan,J.,Manclark,C.R.,Iyengar,R.&Birnbaumer,L.(1984)J.Biol.Chem.259,5871-5886.41.Casey,P.,Graziano,M.P.&Gilman,A.G.(1989)Biochemistry28,611-616.42.Yatani,A.,Imoto,Y.,Codina,J.,Hamilton,S.L.,Brown,A.M.&Birnbaumer,L.(1988)J.Biol.Chem.263,9887-9895.43.Rosenthal,W.,Hescheler,J.,Trautwein,W.&Schultz,G.(1988)FASEBJ.2,2784-2790.44.Skorecki,K.L.,Verkman,A.S.&Ausiello,D.A.(1987)Biochemistry26,639-645.45.KrTpinski,J.,Coussen,F.,Bakalyar,H.A.,Tang,W.-J.,Feinstein,P.G.,Orth,K.,Slaughter,C.,Reed,R.R.&Gilman,A.G.(1989)Science244,1558-1564.WethankDrs.P.Casey,H.Itoh,A.G.Gilman,J.Codina,andL.Birnbaumerforsupplyingasubunits;Drs.M.-Morad,A.-G.Gilman,CellBiology:Kumeetal.