✍ Scribed by Karina Apricò; Philip M. Beart; Duncan Crawford; Ross D. O'Shea
No coin nor oath required. For personal study only.
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.
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