Differential modulation of [3H]TCP binding to the NMDA receptor by L-glutamate and glycine

Abstract At equilibrium (4 h incubation), [ 3 H]TCP (N-(1-[2-thienyl]-cyclohexyl)-3,4-[ 3 H]piperidine) binding to well-washed rat forebrain membranes was enhanced in a concentration-dependent and 2-APV (2-amino-5-phosphonovaleric acid)-sensitive fashion by L-glutamate (EC 50 = 0.2 μ M; maximal effect +280%). L-glutamate (10 μM) increased the affinity of [ 3 H]TCP from 78 to 28 nM, but was without effect on the maximal binding capacity. The enhancing effect of L-glutamate on [ 3 H]TCP binding was potentiated by glycine in a concentration-dependent manner (EC 50 = 50 nM, maximal effect +30% in the presence of 10 μM L-glutamate; EC 50 = 2 μ M, maximal effect +29% in the presence of 0.1 μM L-glutamate). This effect was strychnine-insensitive. Glycine failed to enhance [ 3 H]TCP binding in the presence of 10 μM 2-APV. The glycine effect was due to an increase in affinity (K d = 21 nM in the presence of 10 μM glycine and 10 μM L-glutamate); glycine did not affect the maximal binding capacity. The glycine enhancement of L-glutamate-stimulated [ 3 H]TCP binding was not antagonised by 1 μM strychnine and was mimicked by L-serine and L-alanine but not by GABA, taurine or β-alanine. Kinetic analysis of the glycine and L-glutamate enhancement of [ 3 H]TCP binding indicated that the L-glutamate effect was related to a decrease in the [ 3 H]TCP dissociation rate while the glycine effect was due to an increase in the rate of [ 3 H]TCP association in the presence of L-glutamate.