Functional Reconstitution of a Voltage-Gated Potassium Channel in Giant Unilamellar Vesicles 英文参考文献.docVIP

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Functional Reconstitution of a Voltage-Gated Potassium Channel in Giant Unilamellar Vesicles 英文参考文献.doc

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Functional Reconstitution of a Voltage-Gated Potassium Channel in Giant Unilamellar Vesicles 英文参考文献

FunctionalReconstitutionofaVoltage-GatedPotassium ChannelinGiantUnilamellarVesicles SophieAimon1,JohnManzi1,DanielSchmidt2,JoseAntonioPovedaLarrosa3,PatriciaBassereau1*, GilmanE.S.Toombes1 1Unite′MixtedeRecherche(UMR)168,Physico-ChimieCurie,CentreNationaldelaRechercheScientifique(CNRS),InstitutCurie,CentredeRecherche,Universite′Pierreet MarieCurie,Paris,France,2HowardHughesMedicalInstitute,LaboratoryofMolecularNeurobiologyandBiophysics, RockefellerUniversity,NewYork,NewYork,United StatesofAmerica,3InstitutodeBiolog?′aMolecularyCelular,UniversidadMiguelHerna′ndez, Elche,Spain Abstract Voltage-gatedionchannelsarekeyplayersincellularexcitability.Recentstudiessuggestthattheirbehaviorcandepend stronglyonthemembranelipidcompositionandphysicalstate.Invivostudiesofmembrane/channelandchannel/channel interactions are challenging as membrane properties are actively regulated in living cells, and are difficult to control in experimentalsettings.Wedevelopedamethodtoreconstitutefunctionalvoltage-gatedionchannelsintocell-sizedGiant UnilamellarVesicles(GUVs)inwhichmembranecomposition,tensionandgeometrycanbecontrolled.First,avoltage-gated potassium channel, KvAP, was purified, fluorescently labeled and reconstituted into small proteoliposomes. Small proteoliposomes were then converted into GUVs via electroformation. GUVs could be formed using different lipid compositionsandbufferscontaininglow(5mM)ornear-physiological(100mM)saltconcentrations.Proteinincorporation intoGUVswascharacterizedwithquantitativeconfocalmicroscopy,andtheproteindensityofGUVswascomparabletothe small proteoliposomes from which they were formed. Furthermore, patch-clamp measurements confirmed that the reconstituted channels retained potassium selectivity and voltage-gated activation. GUVs containing functional voltage- gatedionchannelswillallowthestudyofchannelactivity,distributionanddiffusionwhilecontrollingmembranestate,and shouldproveapowerfultoolforunderstandinghowthemembranemodulatescellularexc