93Significanceofpotassiumandchloridemembranecurren

DESALlNATIONELSEVIERDesalination106(1996)43l-434SignificanceofpotassiumandchloridemembranecurrentsinmechanismsofsalttoleranceinCharacwallinaandCharainflataJoseph1.KourieJCSMR,ANU,POBox334,Canberra,ACT2601,AustraliaTel.+61(6)249-2758;Fax[phone]Received7June1995;Accepted5September1995AbstractInvoltageclampexperiments,atwo-pulsevoltageprocedurewasusedtoinvestigatetheioniccurrentsunderlyingtheactionpotential(AP)inCharacoraEEina,afreshwateralgae,andCharainjlata,alesssalt-sensitivealgae.IncomparisonwithC.inJEatu,themembranecurrentsofC.cordinarevealedtwodistinctcharacteristics:theabsenceofa“hump”(i.e.,atransientoutwardK+current,andthepresenceofsuccessionoscillatingpeaks(i.e.,transientinwardCl-currents).InC.inzatathetransientoutwardK”currentorthe“hump”hastheimportantroleofrepolarizingthemembranepotential(V,,*)whichisessentialformaintainingfavourableelectromotiveforceforiontransport.Ontheotherhand,C.corallinaregulatesthestrengthofresponsetoitsenvironmentbyalteringthefrequencyoftheAPtransmittedtothecellinterior.Keywords;Chara;Actionpotential;Salttolerance1.IntroductionTheactionpotential(AP)phenomenonisregardedasamechanismforcoordinationofcellresponsestovariousinternalandexternalstimuli.IncharophytecellsavastamountofinformationonioniccurrentsunderlyingtheAPhasbeenaccumulated.TheAPhasbeenlinkedwithregu-lationofturgorpressureinC.corallina[l]andinAcetabulatiamediterraneaalgae[2].SomespeciesofChavaandLamprothamniumcanbefoundinbrackishsaltwater[3].However,thecharacteristicsandsignificanceoftheAPinrelationtoenvironmentalstresses,e.g.,saltstress,isstillunknown.Itisimportanttobridgethisgapinourknowledgeifwearetounderstandmecha-nismsofsaltsensitivityandtolerance.ThispaperdescribesexperimentsaimedatcomparingtheelectriccurrentunderlyingtheAPinC.inflataandC.corallina.2.MaterialsandMethodsUnlessstatedotherwise,thebasicmethodswereasdescribedbyKourieandFindlay[4].Theartificialpondwater(APW)bathingC.inJEata001I-9164/96/$15.00Copyright01996ElsevierScienceB.V.Allrightsreserved.PIISO01l-9164(96)00142-7432J.1.Kourie/Desalination106(I996)431-434cellscontained(inmM):CaCI,,0.1;KCI,1.O;NaHC03,0.4;HEPES(N-[Z-Hydroxyethyl]pipe-razine-N[2-ethanesulfonic-acid])4.0;withpH7.5adjustedbytheadditionofabout2.6mMNaOH.3,Resultsanddiscussion3.1,Free-runningmembranepotentialVmFig.1showsthetimecourseofl/mandmem-braneresistance(deducedfromthedownwarddeflectioninV,)duringperiodicalcurrentinjec-tioninC.corallina(Fig.1A)andC.inflata(Fig.IS).SpontaneousAPsinthehyperpolarizedrestingpotentialofunclampedC.carallinacellsinthelightareacommonfeatureforthisfreshwateralgae(Fig.IA).ThevmofC.corullinaoscillatesproducingasuccessionofAPs(Fig,IA).Ontheotherhand,C.injataislessexcitablethanC.corallina,andthetransientlight-inducedsingleAPisfollowedbyarapidhyperpolarizationtotherestinglevel(Fig,1B).-1506024681012tImidHowever,C.injZatu.canbemadeexcitablebyblockingKfchannelswithTEA+[S].Inaddition,bothcellsexhibitNaCl-inducedrepetitiveAPs[6,71.3.2.Depotarizingvoltage-clampcurrentsFig.2showsmembranecurrentsduringaseriesoftwo-pulsevoltage-clampstepsinC.corallina(Fig.2A)andC.inJEatu(Fig.2B).V,washeldat-100mVandthenclamped,afterthestandardnegativeprepulseof-200mV,tovariouspotentialsshownontheright.DottedarrowspointtopeaksoftheoscillatingcurrentobservedatVmof-60mV(Fig.2A).Itisevi-dentthatC.corallinalackedtheK’outwardcurrent,l,~,,,~ti.e.,the“hump”whichispresentinCT.inj7ata(Fig.ZB).PatchclampexperimentsonsurgicallyaccessedplasmalemmaofC.coral-hahavefailedtodetectsingleionchannelac-tivitiesthatwouldunderliethetransientdelayedoutwardcomponentI,(,,,,)inC.coraha(see[8]).Anotherdistinctpropertyofthemembrane200Fig.1.TimecourseofV,,Zandmembraneresis-tanceduringperiodicalcurrentinjectioninC.corollinu(A)andC.t(min)inzara(B).Fig.2.Membranecurrentsduringaseriesoftwopulsevoltage.ClampstepsinC.coralha(A)andC.injhtu(B).J.I.Uourie/Desalination106(1996)431-434433CharacorallinaCharainfktaIIIIII104812lb2024t(s)Repolarizai-hnFig.3.A:FlowofioniccurrentsintheequivalentelectricalcircuitforaC.inj7atacell.B:Mem-branecurrentoscillationinC.corallinaandC.inflata.434.I.[.Kourie/Desalination106(I996)431-434currentsofC.corallinawasthepresenceofsuc-cessionofoscillatingpeaks(seedottedarrowsinFig.2A)butnotinC.inj7ata{Fig.2B).3.3.EquivalentelectricalcircuitFig.3Ashowstheflowofioniccurrents(I,,,I,-,,IKandIL)intheequivalentelectricalcircuitforaC.inzatacellillustratingthepassiveelec-tricalpropertiesofthemembrane.Theionicbatteries(EC,,EC,,E,andEL)withelectro-motiveforcesinserieswithconductanceforeachionandtheleak(gc,,gel,gKandgI,)andinpar-allelwithmembranecapacitance(C,J.Thediag-onalarrowsdenotethattheionicconductancesaretime-andvoltage-dependent.UnlikeC.inJlata,C.corallinalackstheK+battery(_I$)andconductance(gK)duringdepolarizingvolt-age-clampsteps.3.4.Significanceqfthe“hump”andcurrentoscillationThephysioIogica1significanceofmembranecurrentoscillationinC.coralhaundersaltstresscouldbehighlightedbycomparingitwithC.iq$ata.UnlikeC.in$‘ata,C,corallinalackedthehump,i.e.,thetransientdelayedoutwardK’currentwhichrepolarizesV,