Subunit-specific roles of glycine-binding domains in activation of NR1/NR3 N-methyl-D-aspartate receptors.

N-Methyl-D-aspartate receptors (NMDARs) composed of NR1 and NR3 subunits differ from other NMDAR subtypes in that they require glycine alone for activation. However, little else is known about the activation mechanism of these receptors. Using NMDAR glycine-site agonists/antagonists in conjunction with functional mutagenesis of the NR1 and NR3 ligand-binding cores, we demonstrate quite surprisingly that agonist binding to NR3 alone is sufficient to activate a significant component of NR1/NR3 receptor currents. Thus, the apo conformation of NR1 in NR1/NR3 receptors is permissive for receptor activation. Agonist-bound NR1 may also contribute to peak NR1/NR3 receptor currents but specifically enables significant NR1/NR3 receptor current decay under the conditions studied here, pre-sumably via a slow component of desensitization. Ligand studies of NR1/NR3 receptors also suggest differential agonist selectivity between NR3 and NR1, as some high-affinity NR1 agonists only minimally activate NR1/NR3 receptors, whereas other NR1 agonists are as potent as glycine. Furthermore, liganded NR3 subunits seem necessary for effective engagement of NR1 in NR1/NR3 receptor activation, suggesting significant interactivity between the two subunits. NR3 subunits thus induce plasticity in NR1 with respect to subunit assembly and ligand binding/channel coupling that is unique among ligand-gated ion channel subunits.

Translationally distinct populations of NMDA receptor subunit NR1 mRNA in the developing rat brain.

The translational activity of the NMDA subunit 1 (NR1) mRNA was examined in the developing rat brain by sucrose gradient fractionation. One translationally-active pool of NR1 mRNA was associated with large polyribosomes (polysomes) over the entire developmental period examined. A second NR1 mRNA pool, approximately half of the NR1 mRNA at post-natal day 4, sedimented only within the two to three ribosome range, indicating that it was translationally blocked during early brain development despite active translation of mRNAs coding for the NR2 subunits of the receptor. At post-natal day 4, both NR1 mRNA pools were distributed throughout the brain and contained similar profiles of NR1 mRNA splice variants, except that NR1-3 appeared to be present only in the translationally-blocked NR1 pool. After post-natal day 8, the translationally-blocked NR1 mRNA pool became progressively active within a background of globally-decreasing brain translational activity.

Translational activity of N-methyl-D-aspartate receptor subunit NR1 mRNA in PC12 cells.

PC12 cells contain NR1 mRNA but lack significant expression of NR1 protein suggesting translational or posttranslational regulation. Translational activity of NR1 mRNA in PC12 cells was examined by sucrose gradient fractionation and by heterologous luciferase NR1 gene expression studies. The cosedimentation and association of NR1 mRNA with large polyribosomes (polysomes) confirmed the translatability of NR1 message in PC12 cells. Possible initiation and/or elongation defects during the translation of NR1 mRNAs were investigated by cyclohexamide treatment. The marked decline in the number of ribosomes associated with NR1 mRNA after prolonged exposure to cyclohexamide suggested that initiation was limiting translation of NR1 mRNA in PC12 cells. Consequently, the effect of the 5' and 3' untranslated regions (UTRs) on translation was examined using fusion constructs consisting of the luciferase coding region fused to either or both the 5' UTR and 3' UTR of NR1. The transfection of PC12 cells with the luciferase NR1-UTR fusion constructs revealed that the 3' UTR of NR1 had a significant inhibitory effect on luciferase expression. In contrast, the 5' UTR of NR1 had no inhibitory effect on mRNA translation in PC12 cells. The results from this study indicate that the translation of NR1 mRNA in PC12 cells may be impeded at initiation and this inhibition may be regulated at least in part through the 3' UTR of NR1.

Excitatory glycine receptors containing the NR3 family of NMDA receptor subunits.

The N-methyl-D-aspartate subtype of glutamate receptor (NMDAR) serves critical functions in physiological and pathological processes in the central nervous system, including neuronal development, plasticity and neurodegeneration. Conventional heteromeric NMDARs composed of NR1 and NR2A-D subunits require dual agonists, glutamate and glycine, for activation. They are also highly permeable to Ca2+, and exhibit voltage-dependent inhibition by Mg2+. Coexpression of NR3A with NR1 and NR2 subunits modulates NMDAR activity. Here we report the cloning and characterization of the final member of the NMDAR family, NR3B, which shares high sequence homology with NR3A. From in situ and immunocytochemical analyses, NR3B is expressed predominantly in motor neurons, whereas NR3A is more widely distributed. Remarkably, when co-expressed in Xenopus oocytes, NR3A or NR3B co-assembles with NR1 to form excitatory glycine receptors that are unaffected by glutamate or NMDA, and inhibited by D-serine, a co-activator of conventional NMDARs. Moreover, NR1/NR3A or -3B receptors form relatively Ca2+-impermeable cation channels that are resistant to Mg2+, MK-801, memantine and competitive antagonists. In cerebrocortical neurons containing NR3 family members, glycine triggers a burst of firing, and membrane patches manifest glycine-responsive single channels that are suppressible by D-serine. By itself, glycine is normally thought of as an inhibitory neurotransmitter. In contrast, these NR1/NR3A or -3B 'NMDARs' constitute a type of excitatory glycine receptor.