title: Constitutive endocytosis and recycling of the neuronal glutamate transporter, excitatory amino acid carrier 1.
abstract: The neuronal glutamate transporter, excitatory amino acid carrier 1 (EAAC1), has a diverse array of physiologic and metabolic functions. There is evidence that there is a relatively large intracellular pool of EAAC1 both in vivo and in vitro, that EAAC1 cycles on and off the plasma membrane, and that EAAC1 cell surface expression can be rapidly regulated by intracellular signals. Despite the possible relevance of EAAC1 trafficking to both physiologic and pathologic processes, the cellular machinery involved has not been defined. In the present study, we found that agents that disrupt clathrin-dependent endocytosis or plasma membrane cholesterol increased steady-state levels of biotinylated EAAC1 in C6 glioma cells and primary neuronal cultures. Acute depletion of cholesterol increased the V(max) for EAAC1-mediated activity and had no effect on Na(+)-dependent glycine transport in the same system. These agents also impaired endocytosis as measured using a reversible biotinylating reagent. Co-expression with dominant-negative variants of dynamin or the clathrin adaptor, epidermal growth factor receptor pathway substrate clone 15, increased the steady-state levels of biotinylated myc-EAAC1. EAAC1 immunoreactivity was found in a subcellular fraction enriched in early endosome antigen 1 (EEA1) isolated by differential centrifugation and partially co-localized with EEA1. Co-expression of a dominant-negative variant of Rab11 (Rab11 S25N) reduced steady-state levels of biotinylated myc-EAAC1 and slowed constitutive delivery of myc-EAAC1 to the plasma membrane. Together, these observations suggest that EAAC1 is constitutively internalized via a clathrin- and dynamin-dependent pathway into early endosomes and that EAAC1 is trafficked back to the cell surface via the endocytic recycling compartment in a Rab11-dependent mechanism. As one defines the machinery required for constitutive trafficking of EAAC1, it may be possible to determine how intracellular signals regulate EAAC1 cell surface expression.
authors.affiliation: Departments of Pediatrics and Pharmacology, Children's Hospital of Philadelphia, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
authors.forename: Marco I
authors.initials: MI
authors.lastname: González
authors.forename: Bala T S
authors.initials: BT
authors.lastname: Susarla
authors.forename: Keith M
authors.initials: KM
authors.lastname: Fournier
authors.forename: Amanda L
authors.initials: AL
authors.lastname: Sheldon
authors.forename: Michael B
authors.initials: MB
authors.lastname: Robinson
mesh_terms: D027322:Amino Acid Transport System X-AG / Q000378:metabolism*; D000818:Animals; D019921:Biotinylation / Q000379:methods; D002478:Cells, Cultured; D004622:Embryo, Mammalian; D004705:Endocytosis / Q000187:drug effects / Q000502:physiology*; D050580:Excitatory Amino Acid Transporter 3 / Q000378:metabolism*; D018698:Glutamic Acid / Q000378:metabolism; D005998:Glycine / Q000378:metabolism; D006624:Hippocampus; D006982:Hypertonic Solutions / Q000494:pharmacology; D051379:Mice; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D021381:Protein Transport / Q000187:drug effects; D051381:Rats; D017207:Rats, Sprague-Dawley; D012964:Sodium / Q000378:metabolism; D013574:Synaptosomes / Q000187:drug effects; D014162:Transfection / Q000379:methods; D047392:beta-Cyclodextrins / Q000494:pharmacology
chemical_list: D027322:Amino Acid Transport System X-AG; D050580:Excitatory Amino Acid Transporter 3; D006982:Hypertonic Solutions; C493399:Slc1a1 protein, mouse; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; D018698:Glutamic Acid; D012964:Sodium; D005998:Glycine