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Binding and transport of [ 3 H](2 S ,4 R )‐ 4‐methylglutamate, a new ligand for glutamate transporters, demonstrate labeling of EAAT1 in cultured murine astrocytes
Author(s) -
Apricò Karina,
Beart Philip M.,
Crawford Duncan,
O'Shea Ross D.
Publication year - 2004
Publication title -
journal of neuroscience research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.72
H-Index - 160
eISSN - 1097-4547
pISSN - 0360-4012
DOI - 10.1002/jnr.20013
Subject(s) - ligand (biochemistry) , glutamate receptor , binding site , biochemistry , transporter , biology , astrocyte , receptor , glycine , microbiology and biotechnology , biophysics , amino acid , endocrinology , gene , central nervous system
Transporters for L ‐glutamate (excitatory amino acid transporters; EAATs), localized to astrocytes, are involved intimately in intermediary metabolism within the brain. Because (2 S ,4 R )‐4‐methylglutamate (4MG) has affinity for glial EAATs, we employed [ 3 H]4MG to define the characteristics of EAATs in cultured murine astrocytes and describe new approaches to analyze EAAT function. Specific binding of [ 3 H]4MG in astrocytic membranes at 4°C represented 90% of total binding. Binding was rapid (apparent t 1/2 ∼7 min) and saturable. Saturation and Scatchard analyses indicated a single binding site (n H = 0.8) with a K d of 6.0 ± 1.5 μM and B max = 9.7 ± 2.9 pmol/mg protein. Binding of [ 3 H]4MG to astrocytic homogenates was Na + ‐dependent and inhibited by K + . Compounds acting at EAATs, such as L ‐glutamate (Glu), D ‐aspartate ( D ‐Asp), L ‐(2 S ,3 S ,4 R )‐2‐(carboxycyclopropyl)glycine and L ‐ trans ‐pyrrolidine‐2,4‐dicarboxylate displaced binding to nonspecific levels. L ‐Serine‐ O ‐sulphate, an EAAT1‐preferring ligand, fully displaced binding of [ 3 H]4MG. In contrast, inhibitors having preferential affinity for EAAT2, L ‐ threo ‐3‐methylglutamate, dihydrokainate, and kainate, were relatively ineffective binding displacers. Agonists and antagonists for Glu receptors failed to significantly inhibit [ 3 H]4MG binding. Studies with [ 3 H] D ‐Asp reinforced evidence that [ 3 H]4MG was binding to EAATs. These data were consistent with Western blot analyses, which indicated abundant expression of EAAT1 but not EAAT2. [ 3 H]4MG was also accumulated rapidly (apparent t 1/2 ∼4 min) into whole astrocytes by a sodium‐ and temperature‐sensitive process (K m of 146 ± 24 μM, V max = 336 ± 27 nmol/mg protein/min), which possessed an EAAT1‐like pharmacologic profile. These findings confirm that 4MG is a substrate for EAAT1 and that the binding assay developed using [ 3 H]4MG can be utilized in various preparations including cultured astrocytes. © 2004 Wiley‐Liss, Inc.