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 [(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 degrees C represented 90% of total binding. Binding was rapid (apparent t(1/2) approximately 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 microM 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-(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 [(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) approximately 4 min) into whole astrocytes by a sodium- and temperature-sensitive process (K(m) of 146 +/- 24 microM, 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.
Department of Pharmacology, Monash University, Clayton, Victoria, Australia.
Karina
K
Apricò
Philip M
PM
Beart
Duncan
D
Crawford
Ross D
RD
O'Shea
D018750:6-Cyano-7-nitroquinoxaline-2,3-dione / Q000494:pharmacology; D027322:Amino Acid Transport System X-AG / Q000378:metabolism*; D000600:Amino Acids, Dicarboxylic / Q000494:pharmacology; D000704:Analysis of Variance; D000818:Animals; D000831:Animals, Newborn; D001253:Astrocytes / Q000187:drug effects / Q000378:metabolism*; D001667:Binding, Competitive; D015153:Blotting, Western / Q000379:methods; D002462:Cell Membrane / Q000187:drug effects; D002478:Cells, Cultured; D026603:D-Aspartic Acid / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D018690:Excitatory Amino Acid Agonists / Q000494:pharmacology; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology; D027341:Excitatory Amino Acid Transporter 1 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D005971:Glutamates / Q000493:pharmacokinetics*; D007608:Kainic Acid / Q000494:pharmacology; D008024:Ligands; D051379:Mice; D011188:Potassium / Q000378:metabolism; D012694:Serine / Q000031:analogs & derivatives* / Q000494:pharmacology; D012964:Sodium / Q000378:metabolism; D013696:Temperature; D014316:Tritium / Q000493:pharmacokinetics
D027322:Amino Acid Transport System X-AG; D000600:Amino Acids, Dicarboxylic; D018690:Excitatory Amino Acid Agonists; D018691:Excitatory Amino Acid Antagonists; D027341:Excitatory Amino Acid Transporter 1; D005971:Glutamates; D008024:Ligands; D014316:Tritium; C046105:4-methylglutamic acid; C035261:(alpha-carboxycyclopropyl)glycine; D012694:Serine; D026603:D-Aspartic Acid; C036040:serine O-sulfate; D018750:6-Cyano-7-nitroquinoxaline-2,3-dione; D012964:Sodium; D011188:Potassium; D007608:Kainic Acid