Expression and Function of Variants of Human Catecholamine Transporters Lacking the Fifth Transmembrane Region Encoded by Exon 6 英文参考文献.docVIP

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Expression and Function of Variants of Human Catecholamine Transporters Lacking the Fifth Transmembrane Region Encoded by Exon 6 英文参考文献.doc

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Expression and Function of Variants of Human Catecholamine Transporters Lacking the Fifth Transmembrane Region Encoded by Exon 6 英文参考文献

ExpressionandFunctionofVariantsofHuman CatecholamineTransportersLackingtheFifth TransmembraneRegionEncodedbyExon6 ChiharuSogawa1,ChiekoMitsuhata2,KeiKumagai-Morioka3,NorioSogawa1,KazumiOhyama4 ,Katsuya Morita3,KatsuyukiKozai2,ToshihiroDohi3,ShigeoKitayama1* 1Department of Dental Pharmacology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan, 2Department of PediatricDentistry,HiroshimaUniversityGraduateSchoolofBiomedicalSciences,Hiroshima,Japan,3DepartmentofDentalPharmacology,HiroshimaUniversityGraduate SchoolofBiomedicalSciences,Hiroshima,Japan,4RIResearchCenter,OkayamaUniversityDentalSchool,Okayama,Japan Abstract Background:Thetransportersfordopamine(DAT)andnorepinephrine(NET)aremembersoftheNa+-andCl2-dependent neurotransmittertransporterfamilySLC6.Thereisalineofevidencethatalternativesplicingresultsinseveralisoformsof neurotransmitter transporters including NET. However, its relevance to the physiology and pathology of the neurotransmitterreuptakesystemhasnotbeenfullyelucidated. Methodology/Principal Findings: We found novel isoforms of human DAT and NET produced by alternative splicing in humanbloodcells(DAT)andplacenta(NET),bothofwhichlackedtheregionencodedbyexon6.RT-PCRanalysesshoweda differenceinexpressionbetweenthefulllength(FL)andtruncatedisoformsinthebrainandperipheraltissues,suggesting tissue-specificalternativesplicing.HeterologousexpressionoftheFLbutnottruncatedisoformsofDATandNETinCOS-7 cellsrevealedtransportactivity.However,immunocytochemistrywithconfocalmicroscopyandacellsurfacebiotinylation assaydemonstratedthatthetruncatedaswellasFLisoformwasexpressedatleastinpartintheplasmamembraneatthe cellsurface,althoughthetruncatedDATwasdistributedtothecellsurfaceslowerthanFLDAT.AspecificantibodytotheC- terminus of DAT labeled the variant but not FL DAT, when cells were not treated with Triton for permeabilization, suggestingtheC-terminusofthevarianttobelocatedextracellulary.Co-expressionoftheFLisoformwi