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The antagonist-bound conformation of the NR2A and NR2B subunits of N-methyl-D-aspartate (NMDA) ionotropic glutamate receptor are modeled using the crystal structure of the DCKA (5,7-dichloro-kynurenic acid)-bound form of the NR1 subunit ligand-binding core (S1S2). Five different competitive NMDA receptor antagonists [(1) DL-AP5; (2) DL-AP7; (3) CGP-37847; (4) CGP 39551; (5) (RS)-CPP] have been docked into both NR2A and NR2B subunits. Experimental studies report NR2A and NR2B subunits having dissimilar interactions and affinities towards the antagonists. However, the molecular mechanism of this difference remains unexplored. The distinctive features in the antagonist's interaction with these two different but closely related (approximately 80% sequence identity at this region) subunits are analyzed from the patterns of their hydrogen bonding. The regions directly involved in the antagonist binding have been classified into seven different interaction sites. Two conserved hydrophilic pockets located at both the S1 and S2 domains are found to be crucial for antagonist binding. The positively charged (Lys) residues present at the second interaction site and the invariant residue (Arg) located at the fourth interaction site are seen to influence ligand binding. The geometry of the binding pockets of NR2A and NR2B subunits have been determined from the distance between the C-alpha atoms in the residues interacting with the ligands. The binding pockets are found to be different for NR2A and NR2B. There are gross dissimilarities in competitive antagonist binding between these two subunits. The binding pocket geometry identified in this study may have the potential for future development of selective antagonists for the NR2A or NR2B subunit.

The ionotropic N-methyl- d-aspartate (NMDA) receptor is of importance in neuronal development, functioning, and degeneration in the mammalian central nervous system. The functional NMDA receptor is a heterotetramer comprising two NR1 and two NR2 or NR3 subunits. We have carried out evolutionary trace (ET) analysis of forty ionotropic glutamate receptor (IGRs) sequences to identify and characterize the residues forming the binding socket. We have also modeled the ligand binding core (S1S2) of NMDA receptor subunits using the recently available crystal structure of NR1 subunit ligand binding core which shares approximately 40% homology with other NMDA receptor subunits. A short molecular dynamics simulation of the glycine-bound form of wild-type and double-mutated (D481N; K483Q) NR1 subunit structure shows considerable RMSD at the hinge region of S1S2 segment, where pore forming transmembrane helices are located in the native receptor. It is suggested that the disruption of domain closure could affect ion-channel activation and thereby lead to perturbations in normal animal behavior. In conclusion, we identified the amino acids that form the ligand-binding pocket in many ionotropic glutamate receptors and studied their hydrogen bonded and nonbonded interaction patterns. Finally, the disruption in the S1S2 domain conformation (of NR1 subunit- crystal structure) has been studied with a short molecular dynamics simulation and correlated with some experimental observations. [figure: see text]. The figure shows the binding mechanism of glutamate with NR2B subunit of the NMDA receptor. Glutamate is shown in cpk, hydrogen bonds in dotted lines and amino acids in blue. The amino acids shown here are within a 4-A radius of the ligand (glutamate).

While it has been well-documented that drugs of abuse such as cocaine can enhance progression of human immunodeficiency virus (HIV)-associated neuropathological disorders, the underlying mechanisms mediating these effects remain poorly understood. The present study was undertaken to examine the effects of cocaine on microglial viability. Herein we demonstrate that exposure of microglial cell line-BV2 or rat primary microglia to exogenous cocaine resulted in decreased cell viability as determined by MTS and TUNEL assays. Microglial toxicity of cocaine was accompanied by an increase in the expression of cleaved caspase-3 as demonstrated by western blot assays. Furthermore, increased microglial toxicity was also associated with a concomitant increase in the production of intracellular reactive oxygen species, an effect that was ameliorated in cells pretreated with NADPH oxidase inhibitor apocynin, thus emphasizing the role of oxidative stress in this process. A novel finding of this study was the involvement of endoplasmic reticulum (ER) signaling mediators such as PERK, Elf2α, and CHOP, which were up regulated in cells exposed to cocaine. Reciprocally, blocking CHOP expression using siRNA ameliorated cocaine-mediated cell death. In conclusion these findings underscore the importance of ER stress in modulating cocaine induced microglial toxicity. Understanding the link between ER stress, oxidative stress and apoptosis could lead to the development of therapeutic strategies targeting cocaine-mediated microglial death/dysfunction.