title: Proximity of transmembrane segments 5 and 8 of the glutamate transporter GLT-1 inferred from paired cysteine mutagenesis.
abstract: BACKGROUND
  GLT-1 is a glial glutamate transporter which maintains low synaptic concentrations of the excitatory neurotransmitter enabling efficient synaptic transmission. Based on the crystal structure of the bacterial homologue Glt(Ph), it has been proposed that the reentrant loop HP2, which connects transmembrane domains (TM) 7 and 8, moves to open and close access to the binding pocket from the extracellular medium. However the conformation change between TM5 and TM8 during the transport cycle is not clear yet. We used paired cysteine mutagenesis in conjunction with treatments with Copper(II)(1,10-Phenanthroline)(3) (CuPh), to verify the predicted proximity of residues located at these structural elements of GLT-1.
  
  
  RESULTS
  To assess the proximity of transmembrane domain (TM) 5 relative to TM8 during transport by the glial glutamate transporter GLT-1/EAAT2, cysteine pairs were introduced at the extracellular ends of these 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 expected to increase the proportion of inward-facing transporters, significantly protected against the inhibition of transport activity of I295C/I463C and G297C/I463C by CuPh. Transport by the double mutants I295C/I463C and G297C/I463C also was inhibited by Cd(2+).
  
  
  CONCLUSIONS
  Our results suggest that TM5 (Ile-295, Gly-297) is in close proximity to TM8 (Ile-463) in the mammalian transporter, and that the spatial relationship between these domains is altered during the transport cycle.
authors.affiliation: China-America Cancer Research Institute, Guangdong Medical College, Dongguan, Guangdong, China.
authors.forename: Xiuping
authors.initials: X
authors.lastname: Zhang
authors.forename: Shaogang
authors.initials: S
authors.lastname: Qu
mesh_terms: D018360:Crystallography, X-Ray; D003545:Cysteine / Q000235:genetics*; D027342:Excitatory Amino Acid Transporter 2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D016296:Mutagenesis; D013438:Sulfhydryl Compounds / Q000378:metabolism
chemical_list: D027342:Excitatory Amino Acid Transporter 2; D013438:Sulfhydryl Compounds; D003545:Cysteine