Proximity of Transmembrane Segments 5 and 8 of the Glutamate Transporter GLT-1 Inferred from Paired Cysteine Mutagenesis 英文参考文献.docVIP

Proximity of Transmembrane Segments 5 and 8 of the Glutamate Transporter GLT-1 Inferred from Paired Cysteine Mutagenesis 英文参考文献.doc

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Proximity of Transmembrane Segments 5 and 8 of the Glutamate Transporter GLT-1 Inferred from Paired Cysteine Mutagenesis 英文参考文献

ProximityofTransmembraneSegments5and8ofthe GlutamateTransporterGLT-1InferredfromPaired CysteineMutagenesis XiupingZhang1,ShaogangQu2* 1China-America Cancer Research Institute, Guangdong Medical College, Dongguan, Guangdong, China, 2Department of Immunology, Southern Medical University, Guangzhou,Guangdong,China Abstract Background: GLT-1 is a glial glutamate transporter which maintains low synaptic concentrations of the excitatory neurotransmitterenablingefficientsynaptictransmission.BasedonthecrystalstructureofthebacterialhomologueGltPh ,it hasbeenproposedthatthereentrantloopHP2,whichconnectstransmembranedomains(TM)7and8,movestoopenand closeaccesstothebindingpocketfromtheextracellularmedium.HowevertheconformationchangebetweenTM5and TM8duringthetransportcycleisnotclearyet.Weusedpairedcysteinemutagenesisinconjunctionwithtreatmentswith Copper(II)(1,10-Phenanthroline)3(CuPh),toverifythepredictedproximityofresidueslocatedatthesestructuralelementsof GLT-1. Methodology/Principal Findings: To assess the proximity of transmembrane domain (TM) 5 relative to TM8 during transportbytheglialglutamatetransporterGLT-1/EAAT2,cysteinepairswereintroducedattheextracellularendsofthese structural elements. A complete inhibition of transport by Copper(II)(1,10-Phenanthroline)3 is observed in the double mutants I295C/I463C and G297C/I463C, but not in the corresponding single mutants. Glutamate and potassium, both expectedtoincreasetheproportionofinward-facingtransporters,significantlyprotectedagainsttheinhibitionoftransport activityofI295C/I463CandG297C/I463CbyCuPh.TransportbythedoublemutantsI295C/I463CandG297C/I463Calsowas 2+ inhibitedbyCd . Conclusions/Significance: Our results suggest that TM5 (Ile-295, Gly-297) is in close proximity to TM8 (Ile-463) in the mammaliantransporter,andthatthespatialrelationshipbetweenthesedomainsisalteredduringthetransportcycle. Citation: Zhang X, Qu S (2011) Proximity of Transmembrane Segments 5 and 8 of the Glutamate Transporter GLT-1 Inferred from Pa

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