Allosteric modulation of [3H]CGP 39653 binding by glycine in rat brain.

D,L-(E)-2-Amino-4-propyl-5-phosphono-3-pentenoic acid (CGP 39653), a new, high-affinity, selective NMDA receptor antagonist, interacts with rat cortical membranes in a saturable way and apparently to a single binding site, with a KD of 10.7 nM and a receptor density of 2.6 pmol/mg of protein. Displacement analysis of [3H]CGP 39653 binding shows a pharmacological profile similar to that reported for another NMDA antagonist, 3-[(+/-)-2-carboxypiperazin-4-yl]propyl-1-phosphonic acid (CPP). Glycine, however, is able to discriminate between the two ligands; in fact, it does not affect [3H]CPP binding but inhibits [3H]CGP 39653 binding in a biphasic way. D-Serine, another agonist at the strychnine-insensitive glycine binding site of the NMDA receptor complex, inhibits [3H]CGP 39653 binding in the same way as glycine, with a potency that correlates with its binding affinity at the glycine site. In addition, 7-chlorokynurenic acid, an antagonist at the glycine site, is able to reverse the displacement of [3H]CGP 39653 by glycine in a dose-dependent manner. Furthermore, the dissociation rate constant of [3H]CGP 39653 is enhanced in the presence of glycine, whereas the presence of NMDA receptor ligands does not modify the rate of dissociation of [3H]CGP 39653 from the receptor. These results indicate that part of the binding of the NMDA antagonist CGP 39653 can be potently modified by glycine through an allosteric mechanism, and suggest the existence of two antagonist preferring NMDA receptor subtypes that are differentially modulated through the glycine binding site.

3-[(+-)-2-carboxypiperazin-4-yl]propyl-1-phosphonic acid recognizes two N-methyl-D-aspartate binding sites in rat cerebral cortex membranes.

Binding of 3-[(+-)-2-carboxypiperazin-4-yl][3H]-propyl-1-phosphonic acid ([3H]CPP), a competitive inhibitor of N-methyl-D-aspartate (NMDA), has been studied in synaptic plasma membranes from rat cerebral cortex. Computer analysis of saturation and homologous displacement isotherms deriving from these plasma membranes indicated the existence of two binding sites: a specific, saturable, high-affinity binding site with a pKD value of 7.53 +/- 0.03 (29.5 nM) and a maximum binding value (Bmax) of 2.25 +/- 0.36 pmol/mg of protein, and a low-affinity site with a KD of approximately 600 nM and a Bmax of 7.0 pmol/mg of protein. It is argued that, in the light of current literature evidence, the low-affinity binding site may represent an agonist-dependent receptor, linked to physiological processes such as neurotransmitter release and channel regulation, whereas the high-affinity binding site may be linked to an antagonist-preferred receptor, for which no function has yet been reported.

Binding of DL-[3H]-alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA) to rat cortex membranes reveals two sites or affinity states.

A method for measuring [3H]-AMPA binding in rat cortex membranes is described. Specific binding was saturable and accounted for 95% of total binding at 5 nM of [3H]-AMPA. Non linear curve fitting of [3H]-AMPA saturation isotherms suggested the presence of two binding sites: the high affinity site showed a pKd of 8.26 +/- 0.07 (Kd = 5.49 nM) and a Bmax of 0.19 +/- 0.03 pmol/mg protein, whereas the low affinity site indicated a pKd of 7.28 +/- 0.05 (Kd = 52 nM) and a Bmax of 1.30 +/- 0.23 pmol/mg protein. The pharmacological profile of [3H]-AMPA binding has been determined by studying a series of compounds in binding displacement experiments: Quisqualate was the most potent inhibitor of [3H]-AMPA binding (IC50 = 9.7 nM), followed by AMPA (19 nM), CNQX, DNQX and L-Glutamate (272-373 nM). Kainate was a moderate displacer (6.2 microM); Ibotenic acid and glycine were very weak inhibitors (74 and 92 microM, respectively). CPP, GAMS and L-Aspartic acid showed IC50-values of over 400 microM and MK-801, DL-AP5 and NMDA were almost inactive at the maximal concentration used in our experiments.