Binding and transport of [3H] (2S,4R)4-methylglutamate, a new ligand for glutamate transporters, demonstrate labeling of EAAT1 in cultured murine astrocytes

Transporters for L-glutamate (excitatory amino acid transporters; EAATs), localized to astrocytes, are involved intimately in intermediary metabolism within the brain. Because (2S,4R)-4-methylglutamate (4MG) has affinity for glial EAATs, we employed [H-3]4MG to define the characteristics of EAATs in cultured murine astrocytes and describe new approaches to analyze EAAT function. Specific binding of [H-3]4MG in astrocytic membranes at 4degreesC represented 90% of total binding. Binding was rapid (apparent t(1/2)similar to7 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 muM and B-max = 9.7 +/- 2.9 pmol/mg protein. Binding of [H-3]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-(2S,3S,4R)-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 [H-3]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 [H-3]4MG binding. Studies with [H-3]D-Asp reinforced evidence that [H-3]4MG was binding to EAATs. These data were consistent with Western blot analyses, which indicated abundant expression of EAAT1 but not EAAT2. [H-3]4MG was also accumulated rapidly (apparent t(1/2) similar to4 min) into whole astrocytes by a sodium- and temperature-sensitive process (K-m of 146 +/- 24 muM, 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 [H-3]4MG can be utilized in various preparations including cultured astrocytes. (C) 2004 Wiley-Liss, Inc.