The time course of the assembly of the N-methyl-D-aspartate receptor was examined in a cell line expressing it under the control of the dexamethasone promoter. These studies suggested a delay between the appearance of the NR1 and NR2A subunits and their stable association as examined by co-immunoprecipitation of NR1 and NR2A. This prompted us to examine the stability and folding of the individual subunits using nonreduced polyacrylamide gels and the sulfhydryl cross-linker BMH. Both studies showed that the NR1 subunit was expressed in a monomer and dimer form, whereas both NR2 and NR3 showed substantial aggregation on both nonreduced gels and after cross-linking. Protein degradation experiments showed that NR1 was relatively stable, whereas NR2 and NR3 were more rapidly degraded. When co-expressed with NR1, NR2 was more stable. Fluorescence recovery after photobleaching experiments showed that, under conditions of reduced ATP, the diffusion rate of NR2 and NR3 in the endoplasmic reticulum was reduced, whereas that of NR1 was unaffected. Together these data show that NR1 folds stably when expressed alone, unlike NR2 and NR3, and provides the substrate for assembly of the N-methyl-D-aspartate receptor.The N-methyl-D-aspartate (NMDA) 2 receptor subtype of the glutamate receptor family are hetero-oligomeric proteins composed of three classes of receptor subunits: NR1, NR2, and NR3. The NR1 subunit is encoded by a single gene, which undergoes extensive splicing to generate eight different splice variants that differ in regional distribution and functional properties (1). The NR2 subunit class consists of four different subtypes, NR2A-NR2D, encoded by four separate but closely related genes (reviewed in Ref. 1). The NR3 subunit class consists of two different subtypes, NR3A and NR3B (2-4). A number of studies of mammalian cell lines either permanently or transiently transfected with the NMDA receptor subunits have indicated that the NR1 subunit alone does not form glycine-glutamate responsive channels and requires the presence of NR2 (5-7).Other studies have shown that the NR1 and NR2 subunits contribute differently to the binding sites of a functional NMDA receptor. The NR1 subunit forms the glycine binding site (8, 9), and the NR2 subunit provides part of the glutamate binding site (10,11). Similar studies on the NR3 subunit family suggested that it could act as a dominant negative subunit that reduced channel conductances when associated with NR1 and NR2 (4, 12). However, several recent studies have suggested that NR3 can combine with NR1 alone to form an excitatory glycine channel in both transfected cells and the central nervous system (13-15). Thus, different combinations of the NMDA receptor subunits must coassemble to form functionally distinct ion channels.Current evidence indicates that the glutamate-activated ion channels are tetrameric. Biochemical and biophysical evidence from studies on ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptors strongly support such a structure (16). These studies also i...

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J. Neurochem. (2010) 114, 1805–1818. Abstract Kainate receptors (KARs) modulate neuronal network activity. The molecular mechanisms that control the assembly and trafficking of KARs are unclear. Here, we examined the role of Q/R editing and subunit composition on KAR subunit assembly and subcellular distribution. The majority of GluK2 subunits undergo editing at the Q/R site in the channel pore loop. Cell surface biotinylation, cross‐linking, Endoglycosidase‐H analysis and gradient separation of KAR subunit assembly states revealed that Q/R editing reduces oligomerization, endoplasmic reticulum (ER) export, plasma membrane expression and stability of homomeric GluK2‐containing KARs. These results indicate that Q/R editing of GluK2 may orchestrate channel subunit composition during KAR assembly in the ER. GluK2/GluK5 heteromers are the most abundant KAR subtype in the brain. While subcellular fractionation of brain tissue confirmed that both GluK2/3 and GluK5 are present in synaptosomes and tightly associated with post‐synaptic density fractions, biochemical analysis revealed that endogenous GluK2/3 subunits show less complete assembly and trafficking compared with GluK5. In transgenic mice, the loss of the key assembly partner GluK2 leads to dramatic reduction in GluK5 expression. These results support the idea that the assembly and intracellular distribution of KARs is determined by RNA editing at the Q/R site and subunit composition.

Competitive N-methyl-D-aspartate receptor (NMDAR) antagonists bind to the GluN2 subunit, of which there are four types (GluN2A-D). We report that some N1-substituted derivatives of cis-piperazine-2,3-dicarboxylic acid display improved relative affinity for GluN2C and GluN2D versus GluN2A and GluN2B. These derivatives also display subtype-selectivity among the more distantly related kainate receptor family. Compounds 18i and (−)-4 were the most potent kainate receptor antagonists and 18i was selective for GluK1 versus GluK2, GluK3 and AMPA receptors. Modeling studies revealed structural features required for activity at GluK1 subunits and suggested that S674 was vital for antagonist activity. Consistent with this hypothesis, replacing the equivalent residue in GluK3 (alanine) with a serine imparts 18i antagonist activity. Antagonists with dual GluN2D and GluK1 antagonist activity may have beneficial effects in various neurological disorders. Consistent with this idea, antagonist 18i (30 mg/Kg i.p.) showed antinociceptive effects in an animal model of mild nerve injury.

Acute injections of melanocortin (MC) agonist and antagonist are highly effective in reducing or increasing food take, respectively. Much less is known about how injection of MC receptor active substances affects metabolism, in particular during long term administration. Here we investigated the effect of 8 days continuous i.c.v. infusion of either MC receptor agonist MTII or the selective MC4 receptor antagonist HS024 on oxygen consumption, food intake and body weight in rats. We observed significant increase in oxygen consumption 2 days after the start of the MTII infusion. However, this increase had disappeared by day 4 of the study. No difference was observed in the oxygen consumption after injection of HS024. MTII substantially decreased the food intake during the first days, but then the feeding recovered and the body weight stabilised at a new level. The immediate effect of the MC receptor agonist on both food intake and metabolism was thus transient, even though the weight loss was maintained. The HS024 treated rats were hyperphagic throughout the test period, continuously gaining weight, resulting in increased fat pads and high leptin levels. This is the first study that describes long term effects of MC receptor agonist and antagonist on metabolism and energy balance.