Two affinity states of N-methyl-D-aspartate recognition sites: modulation by cations

Previous studies have indicated that inorganic and organic cations can markedly affect parameters of the function of the N-methyl-D-aspartate receptor ionophore complex. As these effects may involve modulation of agonist binding, the purpose of our study was to investigate the stimulatory effect of mono- and divalent cations on binding properties of glutamate/N-methyl-D-aspartate recognition sites on the N-methyl-D-aspartate receptor complex, using [3H]CGP 39653 as the specific ligand for these sites. In well-washed membranes from rat brain, [3H]CGP 39653 binding sites were present at two affinity states when assayed at 10 mM HEPES-KOH buffer. About 75% of these sites were in a low-affinity state (Kd = 210 +/- 30 nM) although 25% were in a high-affinity state (Kd = 6.4 +/- 0.4 nM). Addition of mono- or divalent cations to the incubation medium stimulated [3H]CGP 39653 binding, measured at a radioligand concentration of 4 nM. Maximal increases in binding were to approximately 230 and 400% of control, in the presence of mono- and divalent cations, respectively. Values of EC50 for stimulation were 5 to 7 mM for monovalent cations and 0.2 to 0.4 mM for divalent cations. At these concentrations, cations increased the Bmax for the high-affinity population of [3H]CGP 39653 sites and decreased the Bmax for low-affinity ones. These findings suggest that, like spermidine, inorganic cations stimulate binding by converting [3H]CGP 39653 binding sites from the low- to high-affinity state.