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Query: UNIPROT:P06889 (Mol)
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Xenopus oocytes injected with rat brain mRNA were used to identify and characterize the effects of compounds that are antagonists at both the glycine site on N-methyl-D-aspartate (NMDA) receptors and the quisqualate/kainate receptor. Oocytes were voltage-clamped at -60 mV and inward currents were measured at equilibrium following perfusion with agonists and antagonists. Application of 7-chlorokynurenic acid (7-Cl-Kyn) or 6,7-dichloro-3-hydroxy-2-quinoxaline carboxylic acid (6,7-diCl-HQC), each at 15 microM, produced a parallel shift to the right of the glycine concentration-response curve. Schild analysis indicated a KB of 300 nM for 6,7-diCl-HQC and 350 nM for 7-Cl-Kyn. The slopes of the Schild plots were 1.01-1.02 in each case, suggesting that both compounds are competitive glycine antagonists. Both compounds also blocked the receptor mediating kainate-induced inward current. Schild analysis of 6,7-diCl-HQC (KB = 3.0 microM, slope = 0.98) indicated competitive antagonism of kainate currents, but with a potency 10-fold lower than at the glycine site. 7-Cl-Kyn antagonized kainate-evoked currents (KB = 14.1 microM), but the slope of the Schild regression was less than 1 (0.72 +/- 0.11; p less than 0.05). Thus, 7-Cl-kyn was approximately 40-fold more potent at the glycine site than at the receptor mediating kainate currents but is probably not entirely competitive at the latter receptor. Omission of the Cl groups from these antagonists drastically reduced activity at both glycine and kainate sites. 6,7-Dinitro- and 6-cyano-7-nitro-quinoxalinedione were both more potent antagonists of kainate than glycine, but substitution of Cl at the 6-position and especially the 6- and 7-positions increased potency at the glycine site. These results suggest that the glycine coagonist site of the NMDA receptor and the agonist binding site of the quisqualate/kainate receptor have some structural similarity. Halogenated derivatives of quinoxalines and kynurenines should be useful in evaluating the function of the glycine site in synaptic transmission mediated by NMDA receptors. In this regard we found that 7-Cl-kyn (5 and 15 microM) selectively attenuated NMDA receptor-mediated epileptiform bursts in the CA1 region of hippocampal slices perfused with zero-Mg medium, without reducing the amplitude of the primary population spike. This block could be overcome by 300 microM D-serine, which alone did not influence bursting. These results together indicate that the glycine site plays a role in epileptiform bursting mediated by NMDA receptors in adult rat hippocampus.
Mol Pharmacol 1989 Sep
PMID:Selectivity of quinoxalines and kynurenines as antagonists of the glycine site on N-methyl-D-aspartate receptors. 255 Jul 76

Five structural features important for activation of the glycine recognition site on N-methyl-D-aspartate (NMDA) receptors were identified by structure-activity studies of more than 60 glycine analogues in voltage-clamped Xenopus oocytes injected with rat brain mRNA. First, sterically unhindered and ionized carboxyl and amino termini were essential for action at this site. Second, an increase in the interterminal separation by greater than one carbon (e.g., beta-alanine) markedly attenuated activity at this site. Third, activity at the glycine site was stereoselective. The D-isomers of alanine and serine were approximately 20 and 30 times more potent than the L-isomers. Fourth, only small sterically unobtrusive substitutions at the alpha-carbon could be tolerated. alpha-Methyl (D-alanine) and alpha-cyclopropyl (1-amino-cyclopropane carboxylic acid) (ACC) substitutions were effective as agonists but most larger aliphatic and aromatic alpha-carbon substitutions were simply inactive. Glycine, D-alanine, and ACC probably have only a two-point attachment to the receptor. Finally the alpha-carbon substituent of D-serine is envisioned as binding to a third site on the receptor probably via hydrogen bonding of the omega-terminal hydroxyl group. Thus, serine, an hydroxymethyl substitution of glycine, permitted activation of NMDA receptor-mediated currents, whereas isosteric substitutions incapable of hydrogen bonding (e.g., 2-aminobutyric acid) were inactive. Additionally, the position and size of the hydroxyl-containing group is critical for agonist action; D-threonine, DL-homoserine, and hydroxyphenolic substitutions at the alpha-carbon were all inactive. Halogenated analogs of a size comparable to D-serine but capable only of proton acceptance at the omega-terminus (beta-fluoro-D-alanine and beta-chloro-D-alanine) possessed agonist action, whereas an analog capable of only proton donation (1,2-diaminopropionic acid) was inactive. Full concentration-response curves were constructed for those analogs displaying greater than 25% of the effect of glycine when tested at 3 microM. With the exception of (R)-(+)cycloserine and beta-fluoro-D-alanine, all compounds were nearly full agonists and had Hill coefficients not significantly different from unity. The order of relative potency of the active analogs was ACC greater than glycine greater than D-serine greater than D-alanine greater than beta-fluoro-D-alanine greater than (R)-(+)-cycloserine greater than L-serine greater than L-alanine. Molecular modelling of a series of active and inactive analogs with close structural relation to glycine was undertaken. These results were complementary to those data obtained from the electrophysiological investigation.(ABSTRACT TRUNCATED AT 400 WORDS)
Mol Pharmacol 1989 Oct
PMID:Structural requirements for activation of the glycine coagonist site of N-methyl-D-aspartate receptors expressed in Xenopus oocytes. 255 11

A heat-stable factor of low molecular weight that increases the binding of [3H]MK-801 to rat brain membranes in the presence of maximally effective concentrations of L-glutamate and glycine was purified from bovine brain by reverse phase and ion-exchange high pressure liquid chromatography. The stimulatory activity was due to the presence of spermidine in the active fractions. Polyamines including spermine and spermidine are found in high concentrations in mammalian tissue. These compounds increase the affinity of N-methyl-D-aspartate (NMDA) receptors for [3H]MK-801 when assays are carried out in the presence of 100 microM L-glutamate and 100 microM glycine. At concentrations of 1 to 300 microM, a number of di- and triamines, including NH2(CH2)3NH2, NH2(CH2)3NH(CH2)2NH2, and NH2(CH2)3NH(CH2)3NH2, have partial or full agonist-like activity similar to that of spermidine. Other polyamines, including putrescine, cadaverine, NH2(CH2)2NH(CH2)2NH2, and CH3NH(CH2)3NHCH3, at concentrations of 1 to 100 microM, inhibited the binding of [3H]MK-801 in the presence of spermine, L-glutamate, and glycine but not in the presence of only L-glutamate and glycine. It is concluded that these compounds are selective antagonists of the effects of spermine at the NMDA receptor. These results suggest that there may be a polyamine recognition site on the NMDA receptor complex.
Mol Pharmacol 1989 Oct
PMID:Effects of polyamines on the binding of [3H]MK-801 to the N-methyl-D-aspartate receptor: pharmacological evidence for the existence of a polyamine recognition site. 255 12

We have investigated the interactions of polyamines and the N-methyl-D-aspartate (NMDA) receptor antagonist ifenprodil with the binding of [3H]MK801 to the NMDA receptor. Spermine and spermidine but not putrescine substantially increase [3H]MK801 binding to well washed rat brain membranes in the absence or presence of saturating concentrations of glutamate and glycine. Spermine also increased the association and dissociation of [3H]MK801 from its binding site, suggesting that polyamines activate the NMDA receptor in a similar manner to glycine. Ifenprodil inhibited the binding of [3H]MK801 in a biphasic fashion. The high affinity phase of binding (Ki of approximately 15 nM) accounted for 50-60% of total [3H]MK801 binding in the nominal absence of glutamate, glycine, and polyamines or in the presence of 100 microM glutamate. This fraction was reduced to 20% by the addition of 30 microM glycine and could be abolished by the addition of 50 microM spermine. However, ifenprodil apparently did not act by binding to the polyamine recognition site. The low affinity phase (Ki of 20-40 microM) was insensitive to the presence of positive modulators and may represent binding to the Zn2+ regulatory site. Ifenprodil decreased NMDA and glycine-induced Ca2+ influx into cultured rat brain neurons. The potency of ifenprodil suggests that spermine may activate NMDA receptors in vivo. These data indicate that ifenprodil may bind to the NMDA receptor in a state-dependent fashion and preferentially stabilize an inactivated form of the channel.
Mol Pharmacol 1989 Nov
PMID:Ifenprodil is a novel type of N-methyl-D-aspartate receptor antagonist: interaction with polyamines. 255 74

Numerous studies have now demonstrated that a binding site for the psychotomimetic drug phencyclidine (PCP) exists within the receptor channel complex for the excitatory amino acid neurotransmitter glutamate, specifically the glutamate receptor selectively activated by N-methyl-D-aspartate (NMDA). Several lines of evidence support the hypothesis that all PCP receptors in rat brain are associated with the NMDA receptor complex. In the present study, we reexamine this hypothesis. We report that the PCP analog [3H]1-[1-(2-thienyl)cyclohexyl]piperidine [( 3H]TCP) labels two high affinity binding sites in membranes prepared from guinea pig brain site 1 (Kd = 14.1 nM, Bmax = 631 fmol/mg of protein) and site 2 (Kd = 46.5 nM, Bmax = 829 fmol/mg of protein). (+)-5-Methyl-10 11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate bound to site 1 with high affinity (Kl = 3.2 nM) and to site 2 with low affinity (Kl = 5208 nM). The order of potency of drugs for inhibiting [3H]TCP binding to site 1 correlated with their ED50 values for inhibition of NMDA-mediated responses reported in the literature, whereas the order of potency of drugs for inhibiting [3H]TCP binding to site 2 correlated with their ED50 values for inhibition of [3H]dopamine reuptake reported in the literature. Kinetic experiments demonstrated that glutamate, 2-amino-7-phosphonoheptanoic acid, and Mg2+ modulated [3H]TCP binding to site 1 but not site 2. Preincubation of guinea pig striatal membranes with varying concentrations of the high affinity dopamine reuptake inhibitors N-[1-(2-benzo(b)thiophenyl)cyclohexyl]piperidine and 1-[2-[bis(4-fluorophenyl)methoxy]ethyl]-4-[3- phenylpropyl]piperazine caused a wash-resistant inhibition of [3H]TCP binding to site 2 but not site 1. Taken collectively, these data demonstrate the existence of a high affinity PCP binding site associated with the dopamine reuptake carrier and raise the possibility that the therapeutic and psychotomimetic effects of PCP in humans are separable and mediated via different binding sites.
Mol Pharmacol 1989 Dec
PMID:The psychotomimetic drug phencyclidine labels two high affinity binding sites in guinea pig brain: evidence for N-methyl-D-aspartate-coupled and dopamine reuptake carrier-associated phencyclidine binding sites. 255 36

Quantitative pharmacological studies were done to determine the properties of excitatory amino acid receptors expressed in Xenopus oocytes injected with rat brain mRNA. Smooth currents with properties indicative of N-methyl-D-aspartate (NMDA) and quisqualate/kainate receptors were observed in mRNA-injected oocytes. Schild analysis of currents evoked by NMDA indicated that the EAA receptor antagonist D-2-amino-5-phosphonovalerate (D-APV) exerted a competitive block of the oocyte NMDA receptor, because the Schild regression was linear with a slope not significantly different from unity (1.03 +/- 0.025) up to 100 microM D-APV. The pA2 estimated for D-APV antagonism of NMDA currents (5.87 +/- 0.043) was nearly identical to that for D-APV as an L-aspartate antagonist (pA2 = 5.86 +/- 0.073, slope = 0.97 +/- 0.036), suggesting that these two agonists are selective for NMDA receptors in oocytes up to concentrations well above 1 mM. 6-Nitro-7-cyano-quinoxaline-2,3-dione (CNQX) reduced the maximum NMDA response significantly (70% reduction by 15 microM CNQX) but had no effect on the NMDA EC50. CNQX exerted a mixed competitive-noncompetitive block of the glycine site on NMDA receptors; 15 microM CNQX increased the glycine EC50 by 5-fold and reduced the maximum glycine response by 35%. In addition, CNQX exerted a potent and competitive antagonism of currents evoked by kainate. The Schild regression was linear up to 30 microM CNQX with a slope of 1.02 +/- 0.014 and a pA2 of 6.53 +/- 0.029. The block of kainate or NMDA currents by 2 microM CNQX was not voltage dependent. D-APV exerted a weak antagonism of kainate-evoked currents, with a pA2 of 3.39 +/- 0.044, but the slope of the Schild regression was slightly less than 1 (0.90 +/- 0.03). These data demonstrate a clear pharmacological distinction between receptors that mediate the kainate- and NMDA-induced currents and quantify the potency of CNQX and D-APV acting at NMDA/glycine and quisqualate/kainate receptors. The implications of these data for the identification of EAA receptors in oocytes and the classification of neuronal EAA receptors are discussed.
Mol Pharmacol 1989 Mar
PMID:N-methyl-D-aspartate/glycine and quisqualate/kainate receptors expressed in Xenopus oocytes: antagonist pharmacology. 256 33

The interaction of newly described antagonist of the non-NMDA glutamate receptor 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) with the glycine site of the NMDA receptor complex has been investigated. In whole-cell patch recordings from hippocampal neurons maintained in culture, currents induced by N-methyl-D-aspartate (NMDA) were dependent on extracellular glycine. Responses to both NMDA (30 microM) and kainate (20 microM) were reduced by CNQX (10-30 microM). The antagonism by CNQX of NMDA, but not kainate, receptor-mediated responses could be reversed by increasing the concentration of glycine in the external medium. Glycine concentration-response curves constructed in the presence of 30 microM NMDA were shifted to the right by CNQX, suggesting that CNQX was competing with glycine for the glycine binding site. However, even at high concentrations of glycine (300 microM) the maximal NMDA current obtained in the presence of CNQX (10-30 microM) was not restored to control levels. Because CNQX had no effect on responses produced by supramaximal concentrations of NMDA (500 microM) and glycine (300 microM), it is suggested that CNQX also interacts with the NMDA recognition site. The antagonism of currents induced by NMDA was not dependent on the membrane potential, and the rapid onset and offset of the block suggested that there was little or no use dependence. Radioligand binding experiments were performed using [3H]glycine to label the strychnine-insensitive glycine regulatory site of the NMDA receptor complex in guinea pig brain frontal cortex membranes. CNQX displaced [3H]glycine binding in a concentration-dependent manner (IC50 = 5.7 microM). Scatchard analysis of the inhibition showed a decrease in the affinity (increase in Kd) of [3H]glycine binding, but no change in the number of binding sites (Bmax) in the presence of 5 microM CNQX, suggesting a competitive interaction. These data provide evidence that CNQX antagonizes NMDA receptor-mediated responses by competing with glycine for a modulatory site associated with the NMDA receptor complex. Furthermore, the results indicate that CNQX may not be as selective an antagonist for non-NMDA receptors as initially described, although its selectivity will depend on the concentration of the NMDA receptor ligand and may be enhanced by increasing the extracellular concentration of glycine.
Mol Pharmacol 1989 May
PMID:Interaction of 6-cyano-7-nitroquinoxaline-2,3-dione with the N-methyl-D-aspartate receptor-associated glycine binding site. 256 2

Recent studies from our laboratory have provided evidence that multiple states of the phencyclidine (PCP) receptor exist. In addition, several compounds such as PCP and the novel anticonvulsant MK-801 were found to inhibit binding more potently in the presence of Mg2+ and L-glutamate (L-GLU) than when these agents were excluded from the binding assay. In the present study, a number of pharmacological compounds that have been suggested to interact within the N-methyl-D-aspartate (NMDA) receptor complex, including tricyclic antidepressants (TCAs), were examined for their ability to inhibit the binding of [3H]1-[1-(2-thienyl)cyclohexyl]piperidine [( 3H]TCP) in the absence or presence of Mg2+ and L-GLU. The TCAs imipramine, amitriptyline, and opipramol produced shallow inhibition curves in the absence of Mg2+ and L-GLU. Computer analysis of the binding data indicated that a two-component binding model described the data significantly better than a one-component model. In the presence of Mg2+ and L-GLU, the inhibition curves became steeper and were shifted to the right, and computer analysis of the binding data indicated that a one-component model adequately described the binding data. A series of other centrally active compounds, including several antipsychotics and antihistamines, the antiparkinsonian anticholinergic trihexyphenidyl and the antitussive dextromethorphan, were also found to be affected similarly by the inclusion of Mg2+ and L-GLU in the binding assay. Dextrorphan, in contrast to dextromethorphan, inhibited [3H]TCP binding more potently in the presence of Mg2+ and L-GLU. The present results suggest that the compounds that inhibit binding more potently in the absence of Mg2+ and L-GLU are interacting with the PCP receptor in a different manner from that of PCP and MK-801, because these open-channel blockers inhibit [3H]TCP binding more potently in the presence of Mg2+ and L-GLU. The data support previous findings that TCAs interact with the NMDA receptor complex and suggest that the compounds trihexyphenidyl and dextromethorphan, which have been shown to block NMDA-mediated neurotoxicity, may produce their effects through an interaction with the PCP receptor, albeit by a different mechanism from that of open-channel blockers.
Mol Pharmacol 1989 Jul
PMID:Tricyclic antidepressants and dextromethorphan bind with higher affinity to the phencyclidine receptor in the absence of magnesium and L-glutamate. 256 80

The N-methyl-D-aspartate (NMDA)-sensitive glutamate receptors are known to be inhibited by 3-amino-1-hydroxy-2-pyrrolidone (HA-966) and 7-chlorokynurenic acid (Cl-KYN), which act at the glycine-regulated allosteric modulatory center. In this work we show that, in synaptic membranes prepared from rat brain, Cl-KYN and HA-966 inhibit the binding of [3H]glycine. Moreover, Cl-KYN can also completely inhibit the binding of [3H]glutamate to the primary transmitter recognition site for the NMDA receptor, whereas HA-966 only partially reduces this binding. Cl-KYN also abolishes the binding of the NMDA receptor antagonist [3H]3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP). In contrast, HA-966 increases [3H]CPP binding, affecting the affinity but not the maximal number of binding sites. This increase is inhibited by glycine and Cl-KYN. The binding of [3H] (+)-5-methyl-10,11-dihydro-5H-dibenzocyclohepten-5,10-imine maleate (MK-801), used as an index of NMDA receptor activation, is completely inhibited by Cl-KYN but only partially by HA-966. In addition, HA-966, but not Cl-KYN, increases the potency of CPP in inhibiting [3H]MK-801 binding. Our results demonstrate that Cl-KYN and HA-966 differ in their ability to modulate the NMDA receptor, perhaps acting at distinct but overlapping recognition sites. Furthermore, our results suggest that agonist and antagonist recognition sites of the NMDA receptor may be independently regulated by glycine and HA-966, which would result, respectively, in a positive and negative allosteric modulation of the NMDA receptor complex.
Mol Pharmacol 1989 Dec
PMID:Different modes of action of 3-amino-1-hydroxy-2-pyrrolidone (HA-966) and 7-chlorokynurenic acid in the modulation of N-methyl-D-aspartate-sensitive glutamate receptors. 257 20

The N-methyl-D-aspartate (NMDA) receptor consists of a recognition site for NMDA, a cation-selective ion channel, and binding sites for glycine, Zn2+, and phencyclidine-like compounds. In addition, the channel can be blocked by Mg2+. We have studied the NMDA receptor using the potent and specific phencyclidine-like compound [3H]MK-801. Drugs that bind to the NMDA, glycine, Zn2+, and Mg2+ recognition sites profoundly affect both the association and the dissociation rate of [3H]MK-801. NMDA-like agonists, glycine, and Mg2+ all increase the rates of association and dissociation of [3H]MK-801, whereas the NMDA antagonists AP5 and Zn2+ decrease these rates. These data allow the construction of a model of drug interaction at the NMDA receptor that is based on the binding of MK-801 within the NMDA-operated channel. Using this model it is possible to clearly distinguish between drug action at any of the five binding sites proposed.
Mol Pharmacol 1988 Jun
PMID:Multiple sites for the regulation of the N-methyl-D-aspartate receptor. 283 34


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