Characterization of quisqualate recognition sites in rat brain tissue using DL-[3H]alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) and a filtration assay

Abstract

The binding of [3H]AMPA (DL-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid), a ligand for the putative quisqualate excitatory amino acid receptor subtype, was evaluated using centrifugation and filtration receptor binding techniques in rat brain crude synaptosomal membrane preparations. Maximal specific binding of [3H]AMPA occurred in Triton X-100 treated membranes in the presence of the chaotropic agent potassium thiocyanate (KSCN). The effects of KSCN on binding were reversible and optimal at 100 mM. Supernatant obtained from detergent-treated membranes inhibited specific [3H]AMPA and [3H]kainic acid binding, suggesting the presence of an inhibitory agent which was tentatively identified as glutamate. Using centrifugation, saturation analysis revealed two distinct binding sites in both the absence and presence of KSCN. The chaotrope was most effective in increasing binding at the low affinity binding site, enhancing the affinity (Kd) without a concommitant change in the total number of binding sites. Using filtration, a single binding site was detected in Triton-treated membranes. Like the data obtained by centrifugation, KSCN enhanced the affinity of the receptor (Kd value = 10 nM) without altering the number of binding sites (Bmax = 1.2 pmol/mg protein). The rank order of potency of various glutamate analogs in the [3H]AMPA binding assay was quisqualate greater than AMPA greater than L-glutamate greater than kainate greater than D-glutamate, consistent with the labeling of a quisqualate-type excitatory amino acid receptor subtype. L-glutamic acid diethylester, and 2-amino-7-phosphonoheptanoic acid (AP7) were inactive. The present technique provides a rapid, reliable assay for the evaluation of quisqualate-type excitatory amino acid agonists and/or antagonists that may be used to discover more potent and selective agents.