Modulation of Mg 2+ ‐dependent [ 3 H]TCP binding by L ‐glutamate, glycine, and guanine nucleotides in rat cerebral cortex

Biochemical and electrophysiological studies have demonstrated that phencyclidine (PCP) recognition site exists in the ion channel of the N‐methyl‐ D ‐aspartate (NMDA) receptor in ion channel complex. Using an extensively washed rat cortical membrane preparation, the effects of Mg 2+ and guanylylimidodiphosphate (GppNHp) were examined on the binding of [ 3 H]‐N‐[1‐(2‐thienyl)cyclohexyl]‐3, 4‐piperidine ([ 3 H]TCP). Low concentrations of Mg 2+ (EC 50 = 11μM) stimulated [ 3 H]TCP binding under the basal condition and high concentrations of Mg 2+ (IC 50 = 1 mM) inhibited it. In the presence of 10 μM L ‐glutamate and 10 μM glycine, their EC 50 values for Mg 2+ enhancement of [ 3 H]TCP binding were markedly reduced (to 1.9 μM or 8.4 μM), respectively. By contrast, the IC 50 values for Mg 2+ inhibition of [ 3 H]TCP binding were reduced in the presence of L ‐glutamate, but not glycine. Furthermore, a stimulatory effect of Mg 2+ on [ 3 H]TCP binding was additional to the [ 3 H]TCP binding stimulated by a maximally effective concentration of L ‐glutamate (10 μM) or glycine (10 μM). In the kinetic study, 300 μM Mg 2+ produced an increase in the rates of both association and dissociation of [ 3 H]TCP. Similar results were obtained with L ‐glutamate (10 μM) and glycine (10 μM); 10 mM Mg 2+ also caused an acceleration of the association rate but strongly decreased [ 3 H]TCP binding at equilibrium. Compared with [ 3 H]TCP binding under the basal condition, K + (10 mM) alone decreased the maximal binding without producing any change in the association rate; 10 mM K + also significantly decreased Mg 2+ ‐stimulated [ 3 H]TCP binding but caused no change no change in the acceleration of the association rate caused by Mg 2+ . At 100 μM, GppNHp resulted in a rightward shift in the Mg 2+ dose–response curve of [ 3 H]TCP binding to a high affinity site for Mg 2+ under the basal condition and in the presence of glycine. The observations presented here strongly suggest that a Mg 2+ recognition site with high affinity (a high affinity Mg 2+ site) is distinct not only from the L ‐glutamate and glycine binding sites but also from the Mg 2+ recognition site with low affinity located in the ion channel and that the high affinity Mg 2+ site may be associated with a GTP binding site.