Molecular distinction of three N-methyl-D-aspartate-receptor subtypes in situ and developmental receptor maturation demonstrated with the photoaffinity ligand 125I-labeled CGP 55802A.

Abstract

Activation of N-methyl-D-aspartate (NMD)A)receptors is essential for synaptic plasticity in the central nervous system and contributes to neuronal death under various pathological conditions. Although several subunits have been cloned, the structure of NMDA receptors in situ is unresolved. By using a photoreactive antagonist with nanomolar affnity to the NMDA-binding site, three types of receptors were differentiated by their pattern of photoaffinity-labeled METHODS The photoaffinity ligand 125I-labeled CGP 55802A (specific radioactivity 1150 Ci/mmol; 1 Ci = 37 GBq) was synthesized as described elsewhere (R.H., H.A., and C.A., unpublished work). For radioligand binding, rat whole-brain membranes were washed with 100 mM Tris acetate, pH 8.0/0.03% (wt/vol) Triton X-100 and the same buffer without detergent. Aliquots (250 ug ofprotein per assay) were incubated with 1.0 nM '25I-labeled CGP 55802A for 30 min at room temperature in 100 mM Tris acetate, pH 8.0, with the level of nonspecific binding (18% of total binding) being determined in the presence of 2 mM NMDA or 1 mM glutamate. Rapid filtration through GF/C filters (Whatman) was followed by washing twice with 4 ml of ice-cold incubation buffer. For photoaffinity labeling (50 pg of protein per assay) the 30-min incubation was followed by irradiation with UV light (355 nm; 36-W TLD lamp, Philips) for 5 min at a distance of 3 cm. After being washed with 100 mM Tris acetate, pH 8.0, at 40C by centrifugation, the samples were subjected to 6.5% SDS/ PAGE followed by autoradiography (Kodak X-Omat AR film for 2-5 days) and densitometric analysis of the radiotraces (Shimadzu, Kyoto, CS-930). The pattern of photolabeled proteins was the same when Triton X-100 was omitted from the membrane preparation.For autoradiography, cryostat sections, kept in 100 mM Tris acetate, pH 8.0, for 40 min at room temperature, were incubated with 1 nM 125I-labeled CGP 55802A as described above for membranes. For reversible binding the sections were subsequently washed for 20 min in ice-cold incubation buffer (three buffer changes). In the case of photoaffinity labeling sections were exposed to 355-nm light for 5 min, followed by washing for 10 min at room temperature in incubation buffer containing 1 mM glutamate (three buffer changes). The sections were air-dried before exposure to film.Electrophysiological studies were done on transverse hippocampal slices (400 ,um) prepared from male Tif:RAI f(SPF) Abbreviations: NMDA, N-methyl-D-aspartate; Pn, postnatal day n. 8434

The iron-responsive element binding protein: a target for synaptic actions of nitric oxide.

“…However, all of the structures enriched in the IRE-BP correspond to areas with abundant NMDA receptors (43). Because essentially all the IRE-BP localizations we have observed occur in areas of the brain substantially enriched in both NMDA receptors and NOS, physiologic regulation of the IRE-BP by these systems is feasible.…”

The NMDA receptor subunits NR2A and NR2B show histological and ultrastructural localization patterns similar to those of NR1

“…As with the antibody to NR 1, the widespread distribution of NR2A/B antibody is consistent overall with data from NMDA ligand binding studies, and has been discussed in detail for NR 1 antibody . As also noted in that article (see also Monaghan et al, 1993) and demonstrated by pharmacological/binding studies of subunit expression in cell cultures and oocytes (Buller et al, 1993;Marti et al, 1993;Raditsch et al, 1993;Wafford et al, 1993;Yamakura et al, 1993) presence of NMDA receptor complexes made up of NRl in combination with one or more NR2 subunits may account for many of the differences in ligand binding seen in different regions of the brain. Interestingly, higher dendritic staining with NR2A/B antibody, compared to higher cell body staining with our NRl antibody, corresponds with higher binding of NMDA-sensitive @H-glutamate in dendritic zones compared to white matter regions and cell body layers (Monaghan and Cotman, 1985) that is, suggesting a direct relationship between NR2 subunit and ligand-binding site distribution.…”

Regionally different N‐methyl‐D‐aspartate receptors distinguished by ligand binding and quantitative autoradiography of [3H]‐CGP 39653 in rat brain

“…In addition, glycine also inhibited the binding in all areas. The effect was antagonized by the glycine antagonist, 7-CKA, in all regions, indicating that the possibility of negative allosteric modulation of [3H]-CGP 39653 binding, through the activation of the associated strychnine-insensitive glycine site of the NMDA receptor channel complex (Mugnaini et al, 1993) (Lynch et al, 1993;Marti et al, 1993;Laurie & Seeburg, 1994 (Buller et al, 1994) strictly paralleled that of the mRNA encoding for the NR2A subunit (Laurie & Seeburg, 1994 CPP and CGS 19755 which were more in accord with the low affinity site (Fagg et al, 1990;Grimwood et al, 1991 (Monaghan et al, 1988;Buller et al, 1994). It may be noteworthy that in our data, because of the different allosteric modulation, basal [3H]-CGP 39653 binding was already partially inhibited by the endogenous glycine to a greater extent in striatum with respect to the cerebral cortex and the thalamus.…”