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
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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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