title: TM4 of the glutamate transporter GLT-1 experiences substrate-induced motion during the transport cycle.
abstract: Excitatory amino acid transporter 2 (EAAT2), also known as glial glutamate transporter type 1 (GLT-1), plays an important role in maintaining the extracellular glutamate concentrations below neurotoxic levels. The highly conserved TM2 transmembrane domain of GLT-1 maintains a stable position during the transport cycle; however, the effect of the transport cycle on the topology of TM4 in not well established. To further reveal the function of TM4, two cysteine pairs between TM2 and TM4 were introduced using site-directed mutagenesis. A significant decrease of transport activity was observed in the I93C/V241C and I97C/V241C mutants upon application of the oxidative cross-linking reagent, copper (II) (1,10-phenanthroline)3(CuPh), which suggests that a conformational shift is essential for transporter activity. Furthermore, the decrease in activity by CuPh crosslinking was enhanced in external media with glutamate or potassium, which suggests that TM2 and TM4 assume closer proximity in the inward-facing conformation of the transporter. Our results suggest that the TM4 domain of GLT-1, and potentially other glutamate transporters, undergoes a complex conformational shift during substrate translocation, which involves an increase in the proximity of the TM2 and TM4 domains in the inward-facing conformation.
authors.affiliation: Department of Blood Transfusion, The Fifth Affiliated Hospital, Southern Medical University, Guangzhou, Guangdong 510900, China.|Department of Immunology, School of Basic Medical Sciences, Southern Medical University, Guangzhou, Guangdong 510515, China.|Department of Genetic Metabolism, Children's Hospital, Maternal and Child Health Hospital of Guangxi Zhuang Autonomous Region, Nanning, Guangxi 530003, China.
authors.forename: Xiuliang
authors.initials: X
authors.lastname: Rong
authors.affiliation: Department of Neurology, Foshan Hospital of Traditional Chinese Medicine, Guangzhou University of Chinese Medicine, Foshan, Guangdong 528000, China.
authors.forename: Feng
authors.initials: F
authors.lastname: Tan
authors.affiliation: Department of Blood Transfusion, The Fifth Affiliated Hospital, Southern Medical University, Guangzhou, Guangdong 510900, China.|Department of Immunology, School of Basic Medical Sciences, Southern Medical University, Guangzhou, Guangdong 510515, China.
authors.forename: Xiaojuan
authors.initials: X
authors.lastname: Wu
authors.affiliation: Teaching Center of Experimental Medicine, School of Basic Medical Sciences, Southern Medical University, Guangzhou, Guangdong 510515, China.
authors.forename: Xiuping
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Department of Blood Transfusion, The Fifth Affiliated Hospital, Southern Medical University, Guangzhou, Guangdong 510900, China.|Department of Immunology, School of Basic Medical Sciences, Southern Medical University, Guangzhou, Guangdong 510515, China.
authors.forename: Lingli
authors.initials: L
authors.lastname: Lu
authors.affiliation: Department of Thoracic Cardiovascular Surgical, The Fifth Affiliated Hospital, Southern Medical University, Guangzhou, Guangdong 510900, China.
authors.forename: Xiaoming
authors.initials: X
authors.lastname: Zou
authors.affiliation: Department of Blood Transfusion, The Fifth Affiliated Hospital, Southern Medical University, Guangzhou, Guangdong 510900, China.|Department of Immunology, School of Basic Medical Sciences, Southern Medical University, Guangzhou, Guangdong 510515, China.
authors.forename: Shaogang
authors.initials: S
authors.lastname: Qu
mesh_terms: D019943:Amino Acid Substitution; D001693:Biological Transport, Active / Q000502:physiology; D027342:Excitatory Amino Acid Transporter 2; D050491:Glutamate Plasma Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D020125:Mutation, Missense; D000072417:Protein Domains
chemical_list: D027342:Excitatory Amino Acid Transporter 2; D050491:Glutamate Plasma Membrane Transport Proteins; C493397:SLC1A2 protein, human