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Query: UNIPROT:P06889 (Mol)
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The N-methyl-D-aspartate (NMDA) receptor is unique among the ligand-gated ion channels, in that the gating process requires the binding of two independent coagonists, glutamate and glycine. Receptor binding experiments have suggested that the coagonist recognition sites interact with one another in an allosteric manner, and previous work in this laboratory has provided additional functional support in favor of an allosteric coupling; the affinity of glutamate for its recognition site was reduced when a partial agonist, (+)-HA-966, occupied the glycine site, compared with the affinity when glycine itself was bound to the receptor. The present experiments have taken these observations a step further and compare the effects of several glycine site ligands with different affinities and intrinsic activities (determined from equilibrium concentration-response curves) on glutamate off-rate. Thus, the dissociation rate for the decay of glutamate-activated membrane currents in voltage-clamped rat cortical neurons was fastest (160 +/- 28 msec) in the presence of saturating concentrations of (+)-HA-966 and progressively slower in the presence of D-cycloserine (258 +/- 27 msec), aminocyclopropanecarboxylic acid (330 +/- 21 msec), L-alanine (375 +/- 28 msec), and glycine (502 +/- 42 msec). We have also measured the affinities and intrinsic activities of several NMDA receptor ligands and report that a reciprocal interaction exists, such that the off-rate of glycine is influenced by the properties of the agonist occupying the glutamate coagonist site. Thus, the time constant for current decay after a brief exposure to glycine was fastest in the presence of a saturating concentration of cis-2,3-piperidinedicarboxylic acid (449 +/- 26 msec) and progressively slower in the presence of quinolinate (689 +/- 73 msec), NMDA (721 +/- 36 msec), and L-glutamate (1260 +/- 36 msec). The data suggested that the extent of the modulation of one site by the other is related to the intrinsic activity of the agonist, rather than its affinity. Specifically, we suggest that a partial agonist occupying one of the agonist recognition sites produces a conformational change that results in an accelerated off-rate for coagonist dissociation from the receptor; the lower the intrinsic activity, the greater is the effect on coagonist off-rate.
Mol Pharmacol 1994 Dec
PMID:Kinetic study of the interactions between the glutamate and glycine recognition sites on the N-methyl-D-aspartic acid receptor complex. 780 41

The mammalian N-methyl-D-aspartate (NMDA) receptor complex is though to consist of an NR1 subunit in combination with one or more of the four NR2 subunits (A, B, C, and D). When corresponding cDNAs are expressed in Xenopus oocytes, ion channels with the characteristic profile of NMDA receptors are formed. The receptor is unique in requiring two coagonists, glutamate and glycine, for activation of the channel. We have used site-directed mutagenesis to study amino acids in the human NR1 subunit that contribute to the glycine binding site of the NMDA receptor without affecting the agonist site for glutamate. Mutations to D481 and K483 produced receptors with up to 160-fold lower affinities for glycine, as well as other agonists and partial agonists, without affecting maximum current size or the degree of agonist efficacy. The D481A mutation also led to 40-50-fold lower affinities for two structurally diverse glycine site antagonists. From these data we propose that the carboxyl group of this aspartate interacts with the amino moiety of glycine and the equivalent group contained in other agonists and antagonists.
Mol Pharmacol 1995 Feb
PMID:Identification of amino acids in the N-methyl-D-aspartate receptor NR1 subunit that contribute to the glycine binding site. 787 47

Glutamate receptors coupled to polyphosphoinositide (PPI) hydrolysis (metabotropic glutamate receptors, mGluR), are highly efficient during the early stages of postnatal life and are thought to be involved in developmental plasticity. The dramatic decrease with age in mGluR activity suggests the existence of mechanisms that down-regulate this receptor after a certain stage of neuronal maturation. In cultured cerebellar granule neurons grown under conditions that promote the survival and maturation of cells (serum-containing medium with 25 mM K+), enzymatic depletion of extracellular glutamate prevented the age-dependent decrease in mGluR agonist-stimulated PPI hydrolysis that normally occurs after 4 days of maturation in vitro, suggesting that mGluR activity declines as a result of developmental changes affecting homologous desensitization. This was borne out by the observation that glutamate at low concentrations (1-10 microM) readily desensitized mGluR at 7 days but not at 4 days in culture. Furthermore, the critical period during which the high sensitivity to agonist-induced desensitization of mGluR developed coincided with the period when phorbol ester-activated protein kinase C acquired the ability to suppress mGluR activity. The developmental pattern of mGluR agonist-induced PPI hydrolysis was similar in granule cells grown under "trophic" and "nontrophic" conditions (in cultures in 25 mM K+ and in a medium containing "low" K+, in this study, 10 mM, respectively). However, the developmental decline in the response to mGluR stimulation after 4 days in vitro was not prevented in cells grown in 10 mM K+ by the removal of extracellular glutamate; rather, it could be counteracted by treatment with N-methyl-D-aspartate (NMDA) (EC50, approximately 4 microM), which blocked the development of mGluR desensitization. The effect was NMDA receptor mediated and required DNA transcription and protein synthesis. However, NMDA exerted a different effect in cells grown in 25 mM K+, inducing a substantial decrease rather than an increase in mGluR activity. The effect of growth conditions was also examined on mGluR mRNA levels, which were not always correlated with mGluR activity. In general, either increases in the medium K+ concentrations or NMDA supplementation of the cultures resulted in a decrease in mGluR mRNA levels. It is noteworthy that NMDA could also restore mGluR activity after the metabotropic response had reached its peak. This implies that NMDA receptor activation may be involved in the increase in mGluR activity in adult life under conditions that elicit plastic changes in the nervous system.
Mol Pharmacol 1993 Nov
PMID:Mechanisms underlying developmental changes in the expression of metabotropic glutamate receptors in cultured cerebellar granule cells: homologous desensitization and interactive effects involving N-methyl-D-aspartate receptors. 790 31

Accumulating evidence from both radioligand binding and molecular cloning experiments has led to the suggestion that there are heterogeneous populations of N-methyl-D-aspartate (NMDA) receptors. In particular, the NMDA receptor associated with cerebellar neurons has been suggested to be different from that in higher brain regions. With these observations in mind, we considered the possibility that the NMDA receptors on cultured neurons from rat cortex and cerebellum may show differences in their affinity for the coagonists, glutamate and glycine, and the ion channel blocker ketamine. A kinetic approach was used to derive the association and dissociation constants for each of the ligands from their respective macroscopic time constants of binding and unbinding. The constants were assessed electrophysiologically by measuring the onset and decay of whole-cell currents in response to drug applications to voltage-clamped neurons. In addition, differences in coagonist affinity were examined using conventional equilibrium concentration-response curve analysis. These experiments revealed that current relaxations after fast applications of either glutamate or glycine decayed more rapidly in cerebellar neurons, compared with cortical neurons. Thus, assuming two independent binding sites per receptor, the microscopic decay time constants (tau off) for glutamate were 341 +/- 47 (n = 12) and 934 +/- 76 msec (n = 11, p < 0.0001) for granule cells and cortical neurons, respectively. The resulting apparent microscopic dissociation constant (mKd) for glutamate at cerebellar granule cells, calculated from the forward and reverse rate constants, was > 2-fold lower than that for cerebral cortex receptors (496 nM, compared with 251 nM). The difference between the two cell types in the tau off for glycine was more substantial, i.e., 558 +/- 53 (n = 15) and 2214 +/- 125 msec (n = 19, p < 0.0001) for cerebellum and cortex, respectively. Corresponding apparent mKd values for glycine differed by > 4-fold, i.e., 189 nM and 45 nM for cerebellar granule and cortical neurons, respectively. Analysis of data obtained from equilibrium concentration-response curves also revealed differences in coagonist affinity between the two cell populations. The mean mKd values for glutamate at cerebellum and cortical neurons were 1260 nM and 630 nM, respectively, and those for glycine were 316 nM and 63 nM, respectively. No obvious differences were found between the two cell types with respect to the ion channel-blocking kinetics of the dissociative anesthetic ketamine.(ABSTRACT TRUNCATED AT 400 WORDS)
Mol Pharmacol 1993 Dec
PMID:Agonist response kinetics of N-methyl-D-aspartate receptors in neurons cultured from rat cerebral cortex and cerebellum: evidence for receptor heterogeneity. 790 18

In the present study, we have attempted to clarify whether neuroblastoma glioma hybrid NG 108-15 cells (NG cells) possess the NMDA receptor complex using [45Ca2+]influx and [3H]MK-801 binding as functional measures. Glutamate and NMDA dose-dependently increased [45Ca2+]influx and these increases were further enhanced by glycine. Scatchard analysis revealed the presence of a high-affinity binding site for [3H]MK-801 with a KD of 18.8 nM and a Bmax of 0.328 pmol/mg protein. This [3H]MK-801 binding was also increased by NMDA in a dose-dependent manner and this increase was further enhanced by glycine. Both ketamine and MK-801 inhibited glutamate- and NMDA-induced [45Ca2+]influx as well as the increase of [3H]MK-801 binding in a dose-dependent manner. Similarly, Mg2+ and Zn2+ dose-dependently reduced both glutamate-induced [45Ca2+]influx and [3H]MK-801 binding. Spermine, one of the polyamines, showed a biphasic stimulatory effects on glutamate-induced [45Ca2+]influx and [3H]MK-801 binding. These results indicate that NG cells possess a pharmacologically distinct NMDA receptor complex and suggest that these cells may be useful for the analyses on pharmacological and biochemical characteristics of the NMDA receptor complex.
Brain Res Mol Brain Res 1994 Mar
PMID:Presence of N-methyl-D-aspartate (NMDA) receptors in neuroblastoma x glioma hybrid NG108-15 cells-analysis using [45Ca2+]influx and [3H]MK-801 binding as functional measures. 791

An antagonist ligand for the glycine site of the NMDA receptor, [3H]L-689,560, has recently been described. We have investigated the use of this ligand to label NMDA receptors which have been solubilized from rat brain. It has significant advantages over [3H]dizocilpine ([3H]MK-801) for this purpose since (a) it is not inhibited by most detergents, (b) interactions between the glutamate and glycine sites are maintained, and (c) equilibrium binding is rapid and of high affinity (Kd = 8.8 +/- 1.9 nM, n = 4). Nevertheless, precautions must be taken to remove glycine throughout all experimental procedures. In addition we have investigated the ability of NMDA receptors to bind to various lectins and conclude that only N-linked glycosylation is present, consistent with consensus sequences for glycosylation present in cloned subunits of the NMDA receptor. Further binding of the radioligand [3H]L-689,560 was detected both to the solubilized receptor and to receptor immobilized on lectin-agarose, identifying it as an appropriate ligand for use in the characterization of NMDA receptors during purification procedures.
Mol Membr Biol
PMID:Characterization of the solubilized NMDA receptor complex from rat brain with [3H]L-689,560, a glycine site antagonist. 791 9

The powerful psychostimulant and positive reinforcing effects of nicotine have been speculated to be mediated by the dopaminergic neurons of the ventral tegemental area (VTA) and their terminals in the nucleus accumbens. To extend our understanding of nicotine and dopamine interactions, we mapped the pattern of c-fos expression in the striatum as an important marker of some of the earliest changes that occur at gene transcription level. Acute nicotine injections in rats led to Fos expression more prominently in the caudatoputamen than in the nucleus accumbens in a dose-dependent fashion. Fos-reactive cells were more prominent in the central and dorsomedial limbic caudatoputamen than in the dorsolateral sensory-motor striatum. Injections of mecamylamine completely blocked nicotine-induced Fos expression. Injections of the selective dopamine D1 antagonist SCH 23390, but not D2 antagonist YM 09151-2 or Clozapine, a drug with high affinity to D4 receptors, before nicotine injections, completely blocked Fos expression in the striatum. Nicotine induced Fos expression was also blocked completely by the NMDA receptor antagonists MK-801 and CPP. These results suggest that nicotine-induced Fos expression in the striatum is mediated mostly by dopamine D1 receptors and that the Fos expression is also dependent on N-methyl-D-aspartate (NMDA) stimulation.
Brain Res Mol Brain Res 1994 Apr
PMID:Nicotine induced c-fos expression in the striatum is mediated mostly by dopamine D1 receptor and is dependent on NMDA stimulation. 791 1

D1 dopamine (DA) receptor agonists induce the expression of the opioid peptide dynorphin (DYN) in the striatum, an effect accentuated several fold by removing the dopaminergic innervation to the striatum (e.g., by lesioning the DA cell bodies in the substantia nigra [SN]). D1 receptor-mediated effects are thought to involve cAMP and/or phosphoinositides as second messengers. However, it is unclear what third messengers are involved in the regulation of DYN expression. The present experiments evaluated the possible role of two families of immediate-early gene (IEG) proteins, Fos and Jun, in the induction of DYN biosynthesis following repeated treatment with DA agonists. In addition, the role of N-methyl-D-aspartate (NMDA) receptors in modulating DA-induced changes in DYN and IEG protein expression was assessed. Adult male rats received unilateral 6-hydroxydopamine (6-OHDA) or sham lesions of the SN. Following a recovery period, animals were injected twice daily with the DA agonist, apomorphine (APO; 5 mg/kg), for 4 or 7 days. As expected, APO induced DYN biosynthesis, at both the peptide and mRNA level, several fold more in the striatum ipsilateral to the 6-OHDA lesion than in the contralateral control side (or a sham lesioned striatum). These effects appeared to be mediated by D1 receptors since the D1 agonist, SKF 38393 (5 mg/kg), caused the same changes in DYN expression as APO whereas a D2 agonist, quinpirole (1 mg/kg), had no effect. Paralleling the increase in DYN expression, APO also induced the expression of c-Fos and Fos-related antigens (FRA's), in particular a 35 kDa FRA, but had no effect on the expression of various Jun-related IEG proteins (i.e., c-Jun, Jun B, Jun D). Consistent with the notion that Fos and FRA proteins alter transcriptional activity by binding to AP-1 (or AP-1-like) DNA sequences in the promoter regions of target genes, we found that repeated APO treatment caused large increases in AP-1 binding activity in striata ipsilateral to 6-OHDA lesions. These data indicate that repeated activation of D1 receptors increases both the expression of a 35 kDa FRA and AP-1 binding, events which may mediate the large increases in DYN expression in the DA denervated striatum. While co-administration of the NMDA receptor antagonist, MK-801, inhibited APO-induced increases in DYN and Fos/FRA expression in the intact striatum, its only effect in the DA-denervated striatum was a partial (35%) inhibition of the APO-induced increase in DYN-ir concentrations.(ABSTRACT TRUNCATED AT 400 WORDS)
Brain Res Mol Brain Res 1994 May
PMID:Role of a 35 kDa fos-related antigen (FRA) in the long-term induction of striatal dynorphin expression in the 6-hydroxydopamine lesioned rat. 791 58

The non-competitive N-methyl-D-aspartate (NMDA) receptor antagonist dizocilpine maleate (MK-801) stimulates the secretion of adrenocorticotropin hormone (ACTH). As corticotropin-releasing factor (CRF) represents the primary modulator of this secretion, we tested the hypothesis that the ability of MK-801 to activate the hypothalamic-pituitary-adrenal (HPA) axis was modulated through actions at the hypothalamic level that modulate the secretion of CRF. Induction of the immediate-early gene c-fos, as well as of CRF mRNA within the paraventricular nucleus (PVN) of the rat hypothalamus, was examined following the intraperitoneally administration of MK-801 (1 mg/kg). MK-801 markedly increased the expression of Fos-like protein in parvocellular nerve cells of the PVN within 60 min of systemic treatment, and double labeling immunocytochemistry indicated that Fos was primarily localized in CRF-containing neurons of the PVN. MK-801 also significantly increased CRF biosynthesis as detected by in situ hybridization, thus suggesting that c-fos could be involved in the regulation of CRF genes. Taken together, these results suggest that MK-801 stimulates the rat HPA axis probably through the neuronal gene expression of PVN CRF. The significance of these data is discussed in terms of hypothalamic NMDA receptor blockade and subsequent transcriptional regulation of CRF by immediate-early genes.
Brain Res Mol Brain Res 1994 Jul
PMID:Induction of c-fos and CRF mRNA by MK-801 in the parvocellular paraventricular nucleus of the rat hypothalamus. 796 57

Exposure of cultured cerebellar granule neurons to subtoxic concentrations of N-methyl-D-aspartate (NMDA) induces a state of excitoprotection when measured by subsequent exposure to toxic concentrations of glutamate. This NMDA-induced excitoprotective state is prevented by inhibitors of new RNA and protein synthesis. Since the neurotrophic and excitoprotective effects of NMDA in cerebellar granule neurons may involve changes in the expression of the immediate early genes c-fos and c-jun, we measured c-fos and c-jun mRNAs in cerebellar granule neurons after exposure to either toxic concentrations of glutamate or excitoprotective (subtoxic) concentrations of NMDA. Exposure of cerebellar granule neurons to toxic concentrations of glutamate induced a dramatic increase in c-fos and c-jun mRNAs which was not associated with a corresponding increase in c-fos and c-jun proteins as measured immunocytochemically. However, the increase in c-fos and c-jun mRNAs induced by toxic concentrations of glutamate was not altered by preexposing cerebellar granule neurons to NMDA, suggesting that increased expression of c-fos and c-jun mRNAs is not sufficient for glutamate toxicity of these neurons. Preexposure of cerebellar granule neurons to NMDA for 24 h, which induced a maximal excitoprotective state, resulted in a transient increase in c-fos, and to a lesser degree c-jun, mRNAs similar to that induced by toxic concentrations of glutamate. The induction of c-fos, but not that of c-jun, mRNA both by excitoprotective concentrations of NMDA and by neurotoxic concentrations of glutamate was blocked by the non-competitive NMDA receptor antagonist, MK-801.(ABSTRACT TRUNCATED AT 250 WORDS)
Brain Res Mol Brain Res 1994 Mar
PMID:NMDA receptor-mediated excitoprotection of cultured cerebellar granule neurons fails to alter glutamate-induced expression of c-fos and c-jun mRNAs. 801 82


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