Vol. 282, Issue 2, 945-954, 1997

Two Affinity States of N-Methyl-D-Aspartate Recognition Sites: Modulation by Cations

Alexey Mukhin, Elena S. Kovaleva¹ and Edythe D. London

Brain Imaging Section, Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, Baltimore, Maryland (A.M., E.D.L.)

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 (K_d = 210 ± 30 nM) although 25% were in a high-affinity state (K_d = 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 ~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 B_max for the high-affinity population of [³H]CGP 39653 sites and decreased the B_max 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.