title: Molecular determinants of transport stimulation of EAAT2 are located at interface between the trimerization and substrate transport domains.
abstract: Excitatory amino acid transporters (EAATs) regulate glutamatergic signal transmission by clearing extracellular glutamate. Dysfunction of these transporters has been implicated in the pathogenesis of various neurological disorders. Previous studies have shown that venom from the spider Parawixia bistriata and a purified compound (Parawixin1) stimulate EAAT2 activity and protect retinal tissue from ischemic damage. In the present study, the EAAT2 subtype specificity of this compound was explored, employing chimeric proteins between EAAT2 and EAAT3 transporter subtypes and mutants to characterize the structural region targeted by the compound. This identified a critical residue (Histidine-71 in EAAT2 and Serine-45 in EAAT3) in transmembrane domain 2 (TM2) to be important for the selectivity between EAAT2 and EAAT3 and for the activity of the venom. Using the identified residue in TM2 as a structural anchor, several neighboring amino acids within TM5 and TM8 were identified to also be important for the activity of the venom. This structural domain of the transporter lies at the interface of the rigid trimerization domain and the central substrate-binding transport domain. Our studies suggest that the mechanism of glutamate transport enhancement involves an interaction with the transporter that facilitates the movement of the transport domain. We identified a domain (purple star) in the glutamate transporter EAAT2 that is important for transport stimulation through a spider venom, and suggest a mechanism for enhanced transporter function through facilitated substrate translocation (arrow). Because the dysfunction of glutamate transporters is implicated in the pathogenesis of neurological disorders, understanding the mechanisms of enhanced transport could have therapeutic implications.
authors: Department of Pharmacology and Physiology, Drexel University College of Medicine, Philadelphia, Pennsylvania, USA.
authors: Ole V
authors: OV
authors: Mortensen
authors: José L
authors: JL
authors: Liberato
authors: Joaquim
authors: J
authors: Coutinho-Netto
authors: Wagner F
authors: WF
authors: Dos Santos
authors: Andréia C K
authors: AC
authors: Fontana
mesh_terms: D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D019556:COS Cells; D002522:Chlorocebus aethiops; D004305:Dose-Response Relationship, Drug; D027342:Excitatory Amino Acid Transporter 2 / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D018698:Glutamic Acid / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D055503:Protein Multimerization / Q000187:drug effects; D021381:Protein Transport / Q000187:drug effects; D013111:Spider Venoms / Q000737:chemistry; D014118:Toxins, Biological / Q000032:analysis / Q000494:pharmacology; D014162:Transfection
chemical_list: D027342:Excitatory Amino Acid Transporter 2; D013111:Spider Venoms; D014118:Toxins, Biological; D018698:Glutamic Acid
keywords: Parawixia bistriata; excitatory amino acid transporter 2 (EAAT2); glutamate transporter; glutamate uptake; transport enhancement