Comparative studies on binding of 3 different ligands to the N-methyl- d-aspartate recognition domain in brain synaptic membranes treated with Triton X-100

Abstract

Treatment with a low concentration of Triton X-100 almost tripled the binding of [ 3H] d,l-(E)-2-amino-4-propyl-5-phosphono-3-pentenoic acid (CGP 39653), a novel competetive antagonist at an N-methyl- d-aspartate (NMDA)-sensitive subclass of brain excitatory amino acid receptors, in synaptic membranes of the rat brain. The binding linearly increased with increasing protein concentrations of up to 0.4 mg/ml and also increased in proportion to incubation time with a plateau within 60 min after the initiation of incubation at 2°C in Triton-treated membranes. Elevation of incubation temperature from 2°C to 30°C resulted in a marked decrease in the binding at equilibrium by 80%, and a maximal level was obtained within 1 min after the initiation of incubation at 30°C with a gradual decline of up to 10 min. Bound [ 3H]CGP 39653 was rapidly dissociated by the addition of excess unlabeled l-glutamic acid (Glu), and the time required to attain complete dissociation was 60 min at 2°C and 1 min at 30°C, respectively. Among several agonists and antagonists tested, Glu was the most potent displacer of [ 3H]CGP 39653 binding with progressively less potent displacement by d-2-amino-5-phosphonovaleric,(±)-3-(2-carboxypiperain-4-yl)propyl-1-phosphonic (CPP), d-2-amino-7-phosphonoheptanoic, N-methyl- d-aspartic and N-methyl- l-aspartic acids. Agonists at the glycine (Gly) domain on the NMDA receptor ionophore complex invariably inhibited the binding of [ 3H]CGP 39653 without virtually affecting the binding of [ 3H]CPP or [ 3H]Glu in a manner that was sensitive to antagonism by 4 different antagonists at the Gly domain. In contrast, the Gly antagonist 1-hydroxy-3-aminopyrrolidone-2 markedly potentiated [ 3H]CPP binding with the binding of both [ 3H]CGP 39653 and [ 3H]Glu being unaltered. These results suggest that [ 3H]CGP 39653 may predominantly label an antagonist-preferring form of the NMDA domain in a state different from that favorable to labeling by [ 3H]CPP.