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[ 3 H](2 S ,4 R )‐4‐Methylglutamate: a novel ligand for the characterization of glutamate transporters
Author(s) -
Apricó Karina,
Beart Philip M.,
Lawrence Andrew J.,
Crawford Duncan,
O'Shea Ross D.
Publication year - 2001
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1046/j.1471-4159.2001.00337.x
Subject(s) - kainate receptor , binding site , chemistry , receptor , ligand (biochemistry) , serine , glutamate receptor , biochemistry , stereochemistry , ampa receptor , enzyme
[ 3 H](2 S ,4 R )‐4‐Methylglutamate ([ 3 H]4MG), used previously as a ligand for low‐affinity kainate receptors, was employed to establish a binding assay for glutamate transporters (GluTs), as 4MG has also been shown to have affinity for the glial GluTs, GLT1 and GLAST. In rat brain membrane homogenates in the presence of Na + ions at 4°C, specific binding of [ 3 H]4MG was rapid and saturable ( t 1/2 ≈ 15 min), representing > 90% of total binding. Dissociation of [ 3 H]4MG occurred in a biphasic manner, however, saturation studies and Scatchard analysis indicated a single site of binding ( n H = 0.85) and a K d of 6.2 ± 0.8 µ m with a B max of 111.8 ± 23.8 pmol/mg protein. Specific binding of [ 3 H]4MG was Na + ‐dependent and inhibited by K + and HCO 3 − . Pharmacological inhibition with compounds acting at GluTs revealed that Glu, d ‐ and l ‐aspartate, l ‐serine‐ O ‐sulfate and l trans ‐pyrrolidine‐2,4‐dicarboxylate fully displaced specific binding. Drugs having preferential affinity for GLT1, kainate, dihydrokainate and l threo ‐3‐methylglutamate, all inhibited ≈ 40% of specific binding. The inhibition pattern of l ‐serine‐ O ‐sulfate in the presence of a saturating concentration of dihydrokainate was suggestive of [ 3 H]4MG also labelling GLAST. 6‐Cyano‐7‐nitroquinoxaline, a kainate receptor antagonist, and a range of Glu receptor agonists and antagonists failed to significantly inhibit [ 3 H]4MG binding. The pharmacological profile of binding of [ 3 H]4MG resembled that found for [ 3 H] d ‐aspartate, a ligand specific for GluTs, reinforcing the hypothesis that [ 3 H]4MG was labelling GluTs in this assay. Together, these data illustrate the development of an efficient, economic binding assay that is suitable for the characterization of different subtypes of GluTs.