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    Three fluorescent protein voltage sensors exhibit low plasma membrane expression in mammalian cells

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    JournalofNeuroscienceMethods161(200732–38
    Threefluorescentproteinvoltagesensorsexhibitlowplasma
    membraneexpressioninmammaliancells
    B.J.Bakera,∗,H.Leeb,V.A.Pieribonea,c,L.B.Cohena,E.Y.Isacoffb,
    T.Knopfeld,E.K.Kosmidisa,1
    a
    DepartmentofCellularandMolecularPhysiology,YaleUniversitySchoolofMedicine,333CedarSt.,NewHaven,CT06520,USA
    bDepartmentofMolecularandCellBiology,UniversityofCalifornia,Berkeley,CA94720,USA
    cJohnB.PierceLaboratory,NewHaven,CT06520,USA
    dLaboratoryforNeuronalCircuitDynamics,BrainScienceInstitute,RIKEN,2-1Hirosawa,Wako-Shi,Saitama351-0198,Japan
    Received5September2006;receivedinrevisedform27September2006;accepted2October2006
    Abstract
    Threefirst-generationfluorescentproteinvoltagesensitiveprobes(FP-voltagesensorswerecharacterizedinmammaliancells.Flare,aKv1.4variantofFlaSh[SiegelMS,IsacoffEY.Neuron1997;19(October(4:735–41],SPARC[AtakaK,PieriboneVA.BiophysJ2002;82(January(1Pt1:509–16],andVSFP-1[SakaiR,Repunte-CanonigoV,RajCD,KnopfelT.EurJNeurosci2001;13(June(12:2314–18]wereexpressed,imagedandvoltageclampedinHEK293cellsandindissociatedhippocampalneurons.Wewereunabletodetectasignalinresponsetochangesinmembranepotentialafteraveraging16trialswithanyofthethreeconstructs.Usingthehydrophobicvoltagesensitivedye,di8-ANEPPS,asasurfacemarker,confocalanalysesdemonstratedpoorplasmamembraneexpressionforFlare,SPARCandVSFP-1inbothHEK293cellsanddissociatedhippocampalneurons.AlmostalloftheexpressedFP-voltagesensorsresideininternalmembranesinbothcelltypes.Thisinternalexpressiongeneratesabackgroundfluorescencethatincreasesthenoiseintheopticalmeasurement.©2006ElsevierB.V.Allrightsreserved.
    Keywords:Voltagesensor;Fluorescentprotein;di8-ANEPPS;Plasmamembraneexpression
    1.Introduction
    Opticalimagingisaflexiblemethodforstudyingvariouscellularactivities.Organicdyeshavebeendevelopedthatcanfaithfullyreportmanybiologicalvariablesincludingcalciumconcentration,pH,ormembranepotential(Brownetal.,1975;MacDonaldandJobsis,1976;Davilaetal.,1973.Theseopticalprobesenablesimultaneousmeasurementsfrommanylocationsandhavebeenusedtostudythephysiologyofsingleneuronsaswellaslargepopulationsofcells.Forexample,calciumdyestainingoftheolfactorysensoryneuronshasenabledthemap-pingoftheinputsignaltotheolfactorybulbinresponsetoodors(WachowiakandCohen,2001.
    Twomajordrawbacksoftheseorganicdyesaretheindiscrim-inatecellstainingandthelowaccessibilityofthedyetosome
    Correspondingauthor.Tel.:+12037854047.
    E-mailaddress:Bradley.baker@yale.edu(B.J.Baker.
    1AristotleUniversity,SchoolofBiology,Dept.ofZoology,Lab.AnimalPhysiology,Thessaloniki54124,Greece.
    0165-0270/$–seefrontmatter©2006ElsevierB.V.Allrightsreserved.doi:10.1016/j.jneumeth.2006.10.005
    ∗
    celltypes.Tobetterunderstandtheinformationprocessingthatoccursintheolfactorybulb,forexample,onewouldneedtocomparetheneuronaloutputconveyedbythemitralcellstotheinputmapsoftheolfactorysensoryneurons.Wehavebeguntoinvestigategeneticallyencodedvoltagesensitiveprobes(FP-voltagesensorsbecauseinatransgenicanimalageneticallyencodedsensorcouldinprinciplebeexpressedinanycelltypeandwouldhavetheadvantageofstainingonlythecellpopula-tiondeterminedbythespecificityofthepromoterusedtodriveexpression.
    ThefirstFP-voltagesensor,denotedFlaSh,wasobtainedbyinsertingGFPdownstreamoftheporeregionintheDrosophilavoltage-gatedpotassiumchannel,Shaker(SiegelandIsacoff,1997.WhenFlaShwasexpressedinXenopusoocytes,changesinmembranepotentialwerereportedbychangesofitsfluo-rescence.Therateofriseofthefluorescenceresponsevariedfromapproximately10tohundredsofmilliseconds,dependingonwhichvariantofGFPwasused;theywererelativelyslowcomparedtoanactionpotential(Guerreroetal.,2002.AtakaandPieribone(2002insertedGFPintotheratskeletalmus-

    B.J.Bakeretal./JournalofNeuroscienceMethods161(200732–3833
    clevoltage-gatedsodiumchannelandobtainedafasterprobe.Thisconstruct(SPARChasasmallerfluorescencesignalbutfollowschangesinmembranepotentialwithatimeconstantoflessthan1mswhenexpressedinoocytes.Athirdvoltagesensi-tivefluorescentprobe,VSFP-1,hasbeenshowntogiveasignalwhenstablyexpressedinmammalianHEK293cells(Sakaietal.,2001.VSFP-1utilizesfluorescenceresonanceenergytrans-fer(FRETwiththedonorCFPandthereceptorYFPfusedintandemdownstreamoftheS4helixofthepotassiumchannel,Kv1.2.
    ToexaminetheirsuitabilityforuseinatransgenicmousewetestedFlare(aKv1.4-YFPvariantofFlaSh,VSFP-1,andSPARCbytransientexpressioninHEK293cellsanddisso-ciatedhippocampalneurons.Usingwholecellvoltageclamp,wewereunabletodetectanopticalsignal(afteraveraging16trialswithanyoftheconstructsinresponsetochangesinmembranepotentialineithercelltype.Theorganicvolt-agesensitivedye,di8-ANEPPS,wasusedasacontrolintheopticalrecordingmeasurements.di8-ANEPPSalsoservedasacellsurfacemarkertodeterminetheplasmamembraneexpres-sionoftheFP-voltagesensors(Zimmeretal.,2002a,b.AllthreeFP-voltagesensorsexhibitedpredominantlyintracellularstaininginthemammaliancells.Incontrast,thevoltage-gatedpotassiumchannelKv1.4withanN-terminalGFP(Kv1.4-N-GFPandthesodium/potassium/chloridecotransporterwithYFPinsertednearthecarboxy-terminus,NKCC1-YFP,exhib-itedmainlyplasmamembraneexpression.2.Materialandmethods
    2.1.ExpressionofFP-voltagesensorsinmammaliancells
    E18hippocampi(BrainBits,Springfield,ILweredisso-ciatedasdescribedbyBrewerandPrice(1996.DissociatedneuronsandHEK293cellswereplatedontopoly-l-lysinecoatedcoverslips.Transienttransfectionsusinglippofectamine2000(Invitrogenwerecarriedoutfollowingthemanufacturer’sinstructions.TheHEK293celllinestablyexpressingNKCC1-YFPwasagiftfromBiffForbushandMeikePederson.TheplasmidencodinganN-terminalGFPKv1.4protein(Kv1.4-N-GFPwasgiventousbyLevitan(1999.2.2.Patchclampandopticalsignalanalysis
    CoverslipswithHEK293cellsordissociatedhippocam-palneuronswerekeptat37CusingaWarnerinstrumentsmodelSH-27Binlineheaterandstagebathheater.MembranepotentialwascontrolledbyaPatchClampPC-505Bamplifier(WarnerInstruments.Thepipettesolutioncontained120mMK-aspartate,4mMNaCl,4mMMgCl2,1mMCaCl2,10mMEGTA,3mMNa2ATPand5mMHEPESpH7.2.Thebathsolutionconsistedof150mMNaCl,4mMKCl,2mMCaCl2,1mMMgCl2,5mMd-glucose,and5mMHEPESpH7.4.Cellsstainedwith15␮g/mldi8-ANEPPS(Invitrogenwereincubatedfor10–15minatroomtemperature.TheSPARCconstructhasaverylowsodiumconductanceintheabsenceofareducingagent.TheSPARCfluorescentsignalwasnotdependentonthe
    useofareducingagent(AtakaandPieribone,2002.Wedidnotuseareducingagentintheexperimentsreportedhere.
    HEK293cellsordissociatedhippocampalneuronswereimagedonaNikonEclipseE6000FNmicroscopewitha60×,1.0N.A.waterimmersionlensusinga150WXenonarclamp(OptiQuipHighlandMills,NY.SPARCandFlarewereexcitedat480nmwhileVSFP-1(CFPwasexcitedat420nm.SPARC(GFPwasimagedusinga520long-passemissionfilter.Flare(YFPandVSFP-1wereimagedwitha550nmemissionfil-ter.TheobjectiveC-mountimagewasdemagnifiedontotheback-thinnede2vCCD39chipofaNeuroCCD-SM(RedShir-tImaging,LLC,Decatur,GA80pixel×80pixelcamera.TheimagingapparatuswasmountedonaBM-1BenchTopVibra-tionIsolationPlatform(minuskTechnology,Inglewood,CA.Themechanicalshutterintheincidentlightpathwasmountedonaseparatetableanddidnottouchthemicroscope.Aframerateof1kfpswasusedfortheopticalrecordingand4kfpsfortheelectroderecording.Nooff-linetemporalfilteringwasused.SixteentrialswereaveragedforthedatashowninFigs.1and2.Thetracesarethespatialaverageoftheoutputofallofthepixelsreceivinglightfromthecellbody.
    ConfocalmicroscopywasperformedonaZeissLSM-510METAusingaC-Apochromat63×/1.2waterimmersionobjec-tive(Zeiss.Observationofdi8-ANEPPSwasachievedusingtheZeissLP650filterwhiletheFP-voltagesensorswerevisual-izedbypassingthroughtheZeissBP505–530filter.ExcitationoftheFP-voltagesensorsanddi8-ANEPPSwasat488nm.Atleastthreecellspercoverslipwereimagedandtheirpositionsrecordedbeforeadditionof15␮g/mldi8-ANEPPS.Imageswerethentaken2–5minpoststaining.3.Results
    3.1.OpticalsignalsinHEK293cells
    Tomeasurevoltagedependentfluorescencesignals,wholecellvoltageclampwasperformedonHEK293cellsexpress-inganFP-voltagesensor.Asacontrol,wecarriedoutsimilarmeasurementsoncellsstainedwiththeorganicvoltagesensi-tivedye,di8-ANEPPS,orcellsexpressinganN-terminalfusionofGFPandtheKv1.4potassiumchannel(Kv1.4-N-GFP.Themembranepotentialwasheldat−70mVandsubjectedtoa20ms−50mVhyperpolarizingpulsefollowedby+50mVand+100mVdepolarizingpulses(Figs.1and2,blacktrace.ThegreentracesinFigs.1and2showthecurrentsandthefluorescencemeasurementsareinred.TheresultsshowninFigs.1and2arefromtheaverageof16trials.Cellsstainedwithdi8-ANEPPSexhibiteda‘chicken-wire’patternoffluorescence(Fig.1,totallightindicativeofmembranestainingandgaveaneasilymeasuredopticalsignal

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