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MembraneTransport“Pores,PortersandPumps”CH353March4-6,2008Summary•ThermodynamicsandKineticsofMembraneTransport•ClassificationofMembraneTransportProteins–Channels,Porters,PrimaryActiveTransporters•PrimaryActiveTransporters–drivenbyhydrolysisofphosphoanhydridebonds•Porters(secondaryactivetransport&facilitateddiffusion)–drivenbyelectrochemicalpotential•Systemscombiningactivetransportersandporters•Channels(forwaterandions)•Regulationofionchannels–voltageandligandgating–actionpotentialandsynapticfunctionNon-MFSPorters•Na+Ca2+Exchanger(NCX1)–memberofCa2+:cationantiporter(CaCA)family(2.A.19)–antiporterof3Na+for1Ca2+•Na+GlucoseSymporter–memberofsolute:sodiumsymporter(SSS)family(2.A.21)–symportersof1-2Na+for1glucose•HCO3–Cl–Exchangers–membersofanionexchanger(AE)family(2.A.31)–antiportersandsymportersNa+Ca2+Exchanger(NCX1)•PlasmamembraneantiporterregulatingintracellularCa2+incardiacmyocytes•Electrogenicprocess:transports3Na+infor1Ca2+out•LoweraffinityforCa2+thanCa2+ATPasepumps,butfaster•2000Ca2+/secvs.30Ca2+/sec•LoweringCa2+reducesstrengthofheartmusclecontraction•DigoxininhibitsNa+K+ATPaseincreasingintracellularNa+,decreasingefficiencyofNa+Ca2+exchanger,increasingstrengthofheartcontractionNa+Ca2+ExchangerregulatedbycytoplasmicCa2+bindingdomainCBD1onexchangerBlausteinetal.2007,ProcNatlAcadSciUSA104:18349Na+GlucoseSymporters•secondaryactivetransportofglucoseupconcentrationgradientcoupledwithNa+downelectrochemicalgradient(electrogenic)•2humanisoforms:–SGLT1(SLC5A1):highaffinity,lowcapacity•symportof2Na+infor1glucosein•intestinalandrenalabsorptionofglucoseandgalactose•SLC5A1deficiency:glucose-galactosemalabsorption(GGM)–SGLT2(SLC5A2):lowaffinity,highcapacity•symportof1Na+infor1glucosein•renalglucosereabsorption•SLC5A2deficiency:renalglucosuriaGlucoseTransportinIntestinalEpitheliumTightjunction•Apical:SGLT1•Basolateral:GLUT2andNa+K+ATPaseMoleculesabsorbedfromintestinallumenpassthroughepithelialcellsTransportersinepithelialcellsrequirecorrectlocalizationonapicalorbasolateralmembranesEnergeticsofNa+GlucoseTransport•SecondaryactivetransportofglucosecoupledwiththeelectrochemicalgradientofNa+isenergeticallyfavorableNa+out+glucoseout→Na+in+glucosein[Na+]out=145mM[Na+]in=12mM∆y=-50mV∆G(Na+transport)=RTln()+ZF∆y=-6.4kJ/mol+-4.9kJ/mol=-11.3kJ/molC2C1For1molNa+transportedpermolglucose:11.3kJ/mol=RTln()[glucose]in[glucose]out[glucose]in[glucose]out=80.7For2molNa+transportedpermolglucose:22.6kJ/mol=RTln()[glucose]in[glucose]out=6520[glucose]in[glucose]outAtequilibrium:∆G=∆G(Na+transport)+∆G(glucosetransport)=0AnionExchangersHCO3–Cl–Exchangers(Antiporters)•3humanmembersAE1(SLC4A1)(erythrocytes)–bindstocarbonicanhydrase(transportmetabolon)–exchangeofCO2attissuesandlungsAE2(SLC4A2)–intracellularpHregulation;linkedtoNa+H+exchanger–exocrinesecretionAE3(SLC4A3)–isoformsinkidneyandheartFunctionofHCO3–Cl–Exchanger(AE1)RegulationofIntracellularpH•Respiringcellaccumulateexcessacids,e.g.H2CO3•3transportsystemscombinetoregulatepH•Na+H+antiporter(↑pH)–Na+in;H+out•Na+HCO3–Cl–(↑pH)–Na+,HCO3–in;Cl–out•HCO3–Cl–antiporter(↓pH)–HCO3–out;Cl–inIntracellularpHactivatesappropriatetransportsystemIonophoresNon-ribosomallysynthesizedporters•Cycliccompoundsthatcollapsemembraneconcentrationgradients•Diversecomposition(peptides,polyketides,polyethers,tetrolides)•Valinomycin–cyclicpeptideK+uniporter(K+Cs+Na+)•Monesin–cyclicpolyketideNa+:H+antiporter(Na+K+Rb+)•Nigericin–cylicpolyketideK+:H+antiporter(K+Rb+Na+)•Tetronasin–cylicpolyketideCa2+:H+orMg2+:H+antiporter(D-valine–L-lactate–L-valine–D-hydroxyisovalerate)3Valinomycin–K+complexExperimentalMethodsforTransportersModels•Intactcells•Liposomeswithpurifiedorrecombinantproteins•OocyteexpressionofinjectedRNAMeasurements•Membranepotential•Ioncurrent(patchclamp)StudyModelsforTransportProteins•Isolatecellmembranefraction•Purifytransportersfromotherintegralmembraneproteins•Mixsolubilizedtransporterprepwithphospholipids•Removedetergentandformliposomeswithtransporters•UsetransportersinliposomeforexperimentsStudyModelsforTransporters•HarvestoocytesfromXenopuslaevis(oocytesare~1mmdia)•TranscribeinvitroRNAfromacDNAconstruct•MicroinjectRNAintoXenopusoocytes•AllowforexpressionofRNAintoprotein•Measurepropertiesoftransporter[online]MeasurementofTransporterActivityMembranepotentialismeasuredusing:•microelectrodeinsertedintocell,•referenceelectrodeinmedium•potentiometerformeasuringvoltageanddirectionofcurrent•SystemisusedformeasuringactionpotentialsonneuronsandmusclecellsMeasurementofTransporterActivityPatchclampingtechnique•micropipettefilledwithelectrolyteisplacedovertransporteronmembrane•referenceelectrodeinmediumorinsertedincell•measurecurrentrequiredformaintainingconstantamembranepotential•appliedformeasuringtransportofionsMeasurementofTransporterActivity•VariationsonexperimentalsetupforpatchclampingmusclecellorneuronMeasurementofTransporterActivityInterpretingpatchclampingdataa)inside-outpatchesofmusclecellmembraneclampedatvoltageslightlylessthanrestingpotential.Dropsincurrent(pA)indicateopeningofsingleNa+channelsb)patc