title: Mechanisms of substrate transport-induced clustering of a glial glutamate transporter GLT-1 in astroglial-neuronal cultures.
abstract: Glutamate uptake by the Na(+)-dependent glutamate transporter GLT-1, which is predominantly expressed in astrocytes, is crucial for regulating glutamate concentration at the synaptic cleft and achieving proper excitatory neurotransmission. A body of evidence suggests that GLT-1 constitutively traffics between the plasma membrane and endosomes via an endocytosis/recycling pathway, and forms a cluster. Here, we report substrate transport via GLT-1-induced formation of GLT-1 cluster accompanied by intracellular trafficking in rat astroglial-neuronal cultures. We constructed a recombinant adenovirus expressing enhanced green fluorescence protein (EGFP)-tagged GLT-1. Adenoviral infection resulted in the expression of functional GLT-1-EGFP preferentially in astrocytes, partly as clusters. Treatment with glutamate, but not N-methyl-D-aspartate, dramatically increased the number of GLT-1 clusters within 1 h. The estimated EC(50) value of glutamate was 240 microm. In addition, glutamate decreased the cell surface expression and increased the intracellular expression of GLT-1. The GLT-1 clusters were found in early and recycling endosomes and partly in lysosomes, and were inhibited by blockade of endocytotic pathways. Ionotropic and metabotropic glutamate receptor antagonists had no effect on glutamate-induced GLT-1 clustering. The non-transportable glutamate uptake inhibitors (2S,3S)-3-[3-[4-(trifluoromethyl)benzoylamino]benzyloxy]aspartate and dihydrokainate, as well as Na(+)-free conditions, prevented the glutamate-induced GLT-1 clustering, whereas the competitive substrates, aspartate and L-trans-pyrrolidine-2,4-dicarboxylate, induced GLT-1 clustering. Furthermore, the Na(+)/K(+)-ATPase inhibitor, ouabain, and the Na(+) ionophores, gramicidin and monensin, produced GLT-1 clustering. Modulators of intracellular Ca(2+)signaling or membrane depolarization had no effect on GLT-1 clustering. Taken together, these results suggest that Na(+) influx associated with GLT-1 substrate transport triggers the formation of GLT-1 clusters accompanied by intracellular trafficking via endocytotic pathways in astrocytes.
authors.affiliation: Department of Molecular Pharmacology, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan. tnakaga@pharm.kyoto-u.ac.jp
authors.forename: Takayuki
authors.initials: T
authors.lastname: Nakagawa
authors.forename: Yasuto
authors.initials: Y
authors.lastname: Otsubo
authors.forename: Yumi
authors.initials: Y
authors.lastname: Yatani
authors.forename: Hisashi
authors.initials: H
authors.lastname: Shirakawa
authors.forename: Shuji
authors.initials: S
authors.lastname: Kaneko
mesh_terms: D000818:Animals; D001253:Astrocytes / Q000166:cytology / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D018920:Coculture Techniques; D004705:Endocytosis / Q000187:drug effects / Q000502:physiology*; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology; D027342:Excitatory Amino Acid Transporter 2 / Q000235:genetics / Q000378:metabolism*; D018698:Glutamic Acid / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007476:Ionophores / Q000494:pharmacology; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D021381:Protein Transport / Q000187:drug effects / Q000502:physiology; D011506:Proteins / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012964:Sodium / Q000378:metabolism; D015222:Sodium Channels / Q000187:drug effects / Q000378:metabolism*; D022161:Transport Vesicles / Q000378:metabolism / Q000648:ultrastructure; D000097795:ATPase Inhibitory Protein
chemical_list: D018691:Excitatory Amino Acid Antagonists; D027342:Excitatory Amino Acid Transporter 2; D018698:Glutamic Acid; D049452:Green Fluorescent Proteins; D007476:Ionophores; D011506:Proteins; D011993:Recombinant Fusion Proteins; D012964:Sodium; D015222:Sodium Channels; D000097795:ATPase Inhibitory Protein; C485184:enhanced green fluorescent protein