Characterisation of N-methyl-D-aspartate receptor-specific [(3)H]Ifenprodil binding to recombinant human NR1a/NR2B receptors compared with native receptors in rodent brain membranes

We have performed [(3)H]ifenprodil binding experiments under NMDA receptor-specific assay conditions to provide the first detailed characterisation of the pharmacology of the ifenprodil site on NMDA NR1/NR2B receptors, using recombinant human NR1a/NR2B receptors stably expressed in L(tk-) cells, in comparison with rat cortex/hippocampus membranes. [(3)H]Ifenprodil bound to a single, saturable site on both human recombinant NR1a/NR2B receptors and native rat receptors with B:(max) values of 1.83 and 2.45 pmol/mg of protein, respectively, and K:(D) values of 33.5 and 24.8 nM:, respectively. The affinity of various ifenprodil site ligands-eliprodil, (R:(*), R:(*))-4-hydroxy-alpha-(4-hydroxyphenyl)-beta-methyl-4-pehnyl-1-pi per idineethanol [(+/-)-CP-101,606], cis-3-[4-(4-fluorophenyl)-4-hydroxy-1-piperidinyl]-3, 4-dihydro-2H:-1-benzopyran-4,7-diol [(+/-)-CP-283,097], and (R:(*), S:(*))-alpha-(4-hydroxyphenyl)-beta-methyl-4-(phenylmethyl)-1-piperid inepropanol [(+/-)-Ro 25-6981] was very similar for inhibition of [(3)H]ifenprodil binding to recombinant human NR1a/NR2B and native rat receptors, whereas allosteric inhibition of [(3)H]ifenprodil binding by polyamine site ligands (spermine, spermidine, and arcaine) showed approximately twofold lower affinity for recombinant receptors compared with native receptors. Glutamate site ligands were less effective at modulating [(3)H]ifenprodil binding to recombinant NR1a/NR2B receptors compared with native rat receptors. The NMDA receptor-specific [(3)H]ifenprodil binding conditions described were also applied to ex vivo experiments to determine the receptor occupancy of ifenprodil site ligands [ifenprodil, (+/-)-CP-101,606, (+/-)-CP-283,097, and (+/-)-Ro 25-6981] given systemically.     

Actions of butyrophenones and other antipsychotic agents at NMDA receptors: relationship with clinical effects and structural considerations

Haloperidol inhibits NMDA receptors with higher affinity for NMDA receptors composed of NR1/2B compared with NR1/2A. To assess whether the clinical effects of haloperidol and other antipsychotic agents are mediated through this site on NMDA receptors and to examine structure activity relationships at this site, we examined the ability of a variety of drugs with neuroleptic actions to inhibit NMDA receptor function. Many antipsychotic agents inhibit 125I-MK 801 binding to the NMDA receptor with IC50 values in the micromolar range. The rank order of potency for inhibition of binding to adult rat forebrain was trifluperidol (TFP) > clozapine = fluphenazine = reduced haloperidol = spiperone = trifluoperazine = butaclamol > pimozide = risperidone = sulpiride. These findings match the molecular biological specificity of the agents, with trifluperidol having a marked preference for NR1/2B (epsilon2) receptors. Mutations at epsilon2E201, which alter the effects of haloperidol, also decrease the affinity of TFP but not other modulators, showing that the effect of TFP but not other modulators is mediated by this residue of the NMDA receptor. The present results demonstrate that while TFP acts on NMDA receptors in a manner similar to haloperidol, other antipsychotic agents do not share the specific pharmacological properties of this action, suggesting that their clinical mechanism is not mediated by this receptor.     

Pharmacological Characterization of Heterodimeric NMDA Receptors Composed of NR 1a and 2B Subunits: Differences with Receptors Formed from NR 1a and 2A

Abstract: Pharmacological and molecular biological evidence indicates the existence of multiple types of NMDA receptors within the CNS. We have characterized pharmacological properties of receptors assembled from the combination of NR 1a and NR 2B subunits (NR 1a/2B) expressed in transfected cells using both ¹²⁵I-MK-801 binding assays and electrophysiological measures. Binding of ¹²⁵I-MK-801 to cells transfected with NR 1a/2B is saturable with a K _D of 440 p M . The binding is potently inhibited by ketamine, dextromethorphan, phencyclidine, and MK-801 and is stimulated by low concentrations of magnesium. These properties resemble those of native receptors and receptors produced by NR 1a/2A. However, ¹²⁵I-MK-801 binding to membranes from cells transfected with NR 1a/2B is inhibited with high affinity by ifenprodil and is stimulated by spermidine, unlike receptors assembled from NR 1a/2A. NMDA-induced currents measured in cells transfected with either NR 1a/2A or NR 1a/2B have pharmacological properties that correlate well with the binding studies. Currents in cells transfected with NR 1a/2B are potentiated by spermidine and blocked with high affinity by ifenprodil, whereas currents in cells transfected with NR 1a/2A are not enhanced by spermidine and are weakly inhibited by ifenprodil. These data suggest that pharmacological heterogeneity in native NMDA receptors may be explained by combinations of different subunits.     

In vitro characterization of novel NR2B selective NMDA receptor antagonists

N-Methyl-D-aspartate (NMDA) subunit specific receptor antagonism has potential therapeutic application for multiple CNS pathologies. MERCK 1, MERCK 2, and MERCK 3 are novel NR2B subtype selective NMDA receptor antagonists. The affinity and the kinetic mechanism of inhibition by these antagonists and ifenprodil were investigated using the whole-cell configuration of the patch clamp technique, calcium flux, and radioligand binding on a mouse cell line L(tk-) expressing recombinant human heteromeric NMDA receptors consisting of NR1a/NR2B subunit combinations. The rank order of potency, as determined by electrophysiology, was ifenprodil相似文献   

Generation and Characterisation of Stable Cell Lines Expressing Recombinant Human N-Methyl-d-Aspartate Receptor Subtypes

Abstract: Transfection of mouse L(tk-) cells with human N -methyl- d -aspartate (NMDA) receptor subunit cDNAs under the control of a dexamethasone-inducible promoter has been used to generate two stable cell lines expressing NR1a/NR2A receptors and a stable cell line expressing NR1a/NR2B receptors. The cell lines have been characterised by northern and western blot analyses, and the pharmacology of the recombinant receptors determined by radioligand binding techniques. Pharmacological differences were identified between the two NMDA receptor subtypes. The glutamate site antagonist d,l -(ε)-2-[³H]amino-4-propyl-5-phosphono-3-pentanoic acid ([³H]CGP 39653) had high affinity for NR1a/NR2A receptors ( K _D = 3.93 n M ) but did not bind to NR1a/NR2B receptors. Glycine site agonists showed a 2.6–5.4-fold higher affinity for NR1a/NR2B receptors. Data from radioligand binding studies indicated that one of the cell lines, NR1a/NR2A-I, expressed a stoichiometric excess of the NR1a subunit, which may exist as homomeric assemblies. This observation has implications when interpreting data from pharmacological analysis of recombinant receptors, as well as understanding the assembly and control of expression of native NMDA receptors.     

Developmental regulation of NMDA receptor 2B subunit mRNA and ifenprodil binding in the zebra finch anterior forebrain

In passerine songbirds, song learning often is restricted to an early sensitive period and requires the participation of several discrete regions within the anterior forebrain. Activation of N-methyl-D-aspartate (NMDA) receptors is implicated in song learning and in one forebrain song region, the lateral magnocellular nucleus of the anterior neostriatum (IMAN), NMDA receptors decrease in density, their affinity for the antagonist MK-801 increases, and their currents decay more quickly as young male zebra finches lose the ability to imitate new song elements. These developmental changes in NMDA receptor pharmacology and physiology suggest that the subunit composition of NMDA receptors changes developmentally. Here, we have used in situ hybridization and [3H]ifenprodil receptor autoradiography to study the developmental regulation of the NMDA receptor 2B subunit (NR2B) within the anterior forebrain of male zebra finches. NR2B mRNA expression within the IMAN was twice as great in 30-day-old males (early in the sensitive period for song learning) as in adult males, and this developmental decrease in NR2B mRNA expression was mirrored by a decrease in high-affinity (NR2B-associated) [3H]ifenprodil binding within this song region. In another anterior forebrain song region, Area X, NR2B mRNA also declined significantly after 30 days posthatch, but this decline was not accompanied by a significant decrease in [3H]ifenprodil binding. The results are consistent with the hypothesis that developmental changes in NMDA receptor function mediated by regulation of subunit composition contribute to the sensitive period for vocal learning in birds.     

Synaptic activity-dependent developmental regulation of NMDA receptor subunit expression in cultured neocortical neurons

The biophysical properties of NMDA receptors are thought to be critical determinants involved in the regulation of long-term synaptic plasticity during neocortical development. NMDA receptor channel properties are strongly dependent on the subunit composition of heteromeric NMDA receptors. During neocortical development in vivo, the expression of the NMDA receptor 2A (NR2A) subunit is up-regulated at the mRNA and protein level correlating with changes in the kinetic and pharmacological properties of functional NMDA receptors. To investigate the developmental regulation of NMDA receptor subunit expression, we studied NR2 mRNA expression in cultured neocortical neurons. With increasing time in culture, they showed a similar up-regulation of NR2A mRNA expression as described in vivo. As demonstrated by chronic blockade of postsynaptic glutamate receptors in vitro, the regulation of NR2A mRNA was strongly dependent on synaptic activity. In contrast, NR2B mRNA expression was not influenced by activity blockade. Moreover, as shown pharmacologically, the regulation of NR2A mRNA expression was mediated by postsynaptic Ca(2+) influx through both NMDA receptors and L-type Ca(2+) channels. It is interesting that even relatively weak expression of NR2A mRNA was correlated with clearly reduced sensitivity of NMDA receptor-mediated whole-cell currents against the NR2B subunit-specific antagonist ifenprodil. Developmental changes in the expression of NR1 mRNA splice variants were also strongly dependent on synaptic activity and thus might, in addition to regulation of NR2 subunit expression, contribute to developmental changes in the properties of functional NMDA receptors. In summary, our results demonstrate that synaptic activity is a key factor in the regulation of NMDA receptor subunit expression during neocortical development.     

Four residues of the extracellular N-terminal domain of the NR2A subunit control high-affinity Zn2+ binding to NMDA receptors

NMDA receptors are allosterically inhibited by Zn2+ ions in a voltage-independent manner. The apparent affinity for Zn2+ of the heteromeric NMDA receptors is determined by the subtype of NR2 subunit expressed, with NR2A-containing receptors being the most sensitive (IC50, approximately 20 nM) and NR2C-containing receptors being the least sensitive (IC50, approximately 30 microM). Using chimeras constructed from these two NR2 subtypes, we show that the N-terminal LIVBP-like domain of the NR2A subunit controls the high-affinity Zn2+ inhibition. Mutations at four residues in this domain markedly reduce Zn2+ affinity (by up to >500-fold) without affecting either receptor activation by glutamate and glycine or inhibition by extracellular protons and Ni2+ ions, indicating that these residues most likely participate in high-affinity Zn2+ binding.     

Modulation of triheteromeric NMDA receptors by N-terminal domain ligands

NMDA receptors (NMDARs) are heteromeric assemblies of NR1 and NR2(A-D) subunits with properties heavily influenced by the type of NR2 subunit incorporated. While NMDARs with only one type of NR2 subunit have been extensively characterized, little is known about receptors containing two different NR2 subunits, despite compelling evidence that such triheteromeric receptors exist in vivo. We used a point-mutation approach that allows isolation of recombinant triheteromeric NMDARs possessing two different NR2 N-terminal domains (NTDs). We show that in receptors associating the NR2A-NTD (sensing nanomolar Zn) and the NR2B-NTD (sensing ifenprodil), each NTD binding site retains selective high affinity for its ligand. However, each ligand produces only partial inhibition, and maximal inhibition requires occupancy of both NR2-NTDs by their respective ligands. Similarly, NR1/2A/2C receptors are inhibited by zinc with high potency but low efficacy. Therefore, interactions between homologous N-terminal domains determine the unique pharmacological properties of triheteromeric NMDARs.     

Effects of Acute and Chronic Ethanol Exposure on Heteromeric N-Methyl-d-Aspartate Receptors Expressed in HEK 293 Cells

Abstract: Ion flux through native N-methyl-d -aspartate (NMDA) receptors is inhibited by behaviorally relevant concentrations of ethanol (10–100 mM) in a variety of neuronal preparations. However, in animal tissues, it is often difficult to determine accurately which NMDA receptor subunits are responsible for the observed effect. In this study, human embryonic kidney 293 (HEK 293) cells normally devoid of NMDA receptors were transiently transfected with cDNA expression plasmids coding for specific rat NMDA receptor subunits. Brief application of an NMDA/glycine solution to cells markedly increased intracellular calcium in cells transfected with NR1/NR2A, NR1/NR2B, or NR1/NR2A/NR2B as measured by fura-2 calcium imaging. This increase was both NMDA- and glycine-dependent and was inhibited by competitive and noncompetitive NMDA antagonists, including 2-amino-5-phosphopentanoic acid and MK-801. The NR2B-selective antagonist ifenprodil inhibited responses in cells transfected with NR1/NR2B or NR1/NR2A/NR2B, but not NR1/NR2A subunits. Increasing the transfection ratio of NR2B versus NR2A subunit in NR1/NR2A/NR2B-transfected cells greatly increased their ifenprodil sensitivity. Acute exposure to ethanol (25–100 mM) inhibited the NMDA-mediated increase in intracellular calcium in a dose-dependent manner without affecting basal calcium concentrations. There were no statistically significant differences in ethanol's potency or maximal inhibition between any of the subunit combinations tested. HEK 293 cells transfected with NR1/NR2A/NR2B subunits showed an enhanced sensitivity to ifenprodil following a 24-h exposure to concentrations of ethanol of 50 mM and greater. The enhanced ifenprodil sensitivity following ethanol exposure was not associated with changes in NR1, NR2A, or NR2B immunoreactivity. In contrast to results obtained in transfected HEK 293 cells, no effect of chronic ethanol was observed in oocytes expressing NR1/NR2A/NR2B subunits. These results demonstrate that recombinant NMDA receptors expressed in HEK 293 cells form functional receptors that, like native receptors, are sensitive to modulation by both acute and chronic ethanol treatment.     

NMDA受体在焦虑症发病机制的研究进展

伴随社会生活和工作压力的增大,常见精神类疾病焦虑症的发病率逐年攀升。焦虑症的发病机制非常复杂,迄今尚未完全阐明。本文概述了焦虑症发病机制与NMDA受体不同亚型的关系。NMDA受体主要广泛分布于脑、脊髓和周围神经系统。NR1广泛分布于中枢神经,在NR2D亚基敲除小鼠中,NR1和NR2D的相互影响可能参与了焦虑样行为。NR2A与NR2B是NMDA受体的两个重要亚基,NR2B的高选择性拮抗剂艾芬地尔在小鼠的高架十字迷宫实验中发挥了抗焦虑功效。将小鼠全脑的NR2C基因用NR2B替代之后,1月龄变异小鼠的高架十字迷宫实验显示有明显的非条件性焦虑行为,表明NR2B和NR2C均可能参与焦虑的发生。因此,深入阐明调控NMDA受体亚基组成的确切作用机制,将有助于探索焦虑症潜在治疗靶点的发现,并针对性地开展新药的研发。     

Modulation of 45Ca2+ Influx into Cells Stably Expressing Recombinant Human NMDA Receptors by Ligands Acting at Distinct Recognition Sites

Abstract: A ⁴⁵Ca²⁺ influx assay has been used to investigate the pharmacology of stably expressed recombinant human NR1a/NR2A and NR1a/NR2B N -methyl- d -aspartate (NMDA) receptors. Inhibition of glutamate-stimulated ⁴⁵Ca²⁺ influx by six glycine-site antagonists and inhibition of glycine-stimulated ⁴⁵Ca²⁺ influx by five glutamate-site antagonists revealed no significant differences between affinity values obtained for NR1a/NR2A and NR1a/NR2B receptors. The polyamine site agonist spermine showed differential modulation of glutamate- and glycine-stimulated ⁴⁵Ca²⁺ influx for recombinant NMDA receptors, inhibiting and stimulating ⁴⁵Ca²⁺ influx into cells expressing NR1a/NR2A receptors (IC₅₀ = 408 µ M ) and NR1a/NR2B receptors (EC₅₀ = 37.3 µ M ), respectively. The antagonist ifenprodil was selective for NR1a/NR2B receptors (IC₅₀ = 0.099 µ M ) compared with NR1a/NR2A receptors (IC₅₀ = 164 µ M ). The effects of putative polyamine site antagonists, redox agents, ethanol, and Mg²⁺ and Zn²⁺ ions were also compared between NR1a/NR2A and NR1a/NR2B receptors. This study demonstrates the use of ⁴⁵Ca²⁺ influx as a method for investigating the pharmacology of the numerous modulatory sites that regulate the function of recombinant human NMDA receptors stably expressed in L(tk-) cells.     

Neurosteroids allosterically modulate the ion pore of the NMDA receptor consisting of NR1/NR2B but not NR1/NR2A

Neurosteroids are endogenously derived compounds, mediating rapid effects in the central nervous system. They participate in vital processes, including memory and learning, neuroplasticity, and neuroprotection in Alzheimer’s disease. However, the mechanisms behind those effects remain to be elucidated. The neurosteroids pregnenolone sulphate (PS) and pregnanolone sulphate (3α5βS) have recently been shown to allosterically alter the NMDA receptor in nanomolar concentrations. Those studies featured ifenprodil, which is a dirty drug, with affinity to many targets. In this study we compare the NMDA receptors in the hippocampus to recombinant NMDA receptors, using [³H]-MK-801 as radioligand. The results show that neurosteroids modulate the ifenprodil binding kinetics in a narrow concentration interval, addressing it to the NR2B subunit, since no effects were recorded at recombinant NR1/NR2A receptors. The effects were also seen as changes in the manner ifenprodil displaced or induced the dissociation of [³H]-MK-801. It indicates that the neurosteroidal effects indeed alter the ion pore of the NMDA receptor, why it is reasonable to believe that these findings have physiological relevance.     

Exposure to an enriched environment selectively increases the functional response of the pre-synaptic NMDA receptors which modulate noradrenaline release in mouse hippocampus

We evaluated the impact of environmental training on the functions of pre-synaptic glutamatergic NMDA and α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) and nicotinic receptors expressed by hippocampal noradrenergic nerve terminals. Synaptosomes isolated from the hippocampi of mice housed in enriched (EE) or standard (SE) environment were labeled with [³H]noradrenaline ([³H]NA) and tritium release was monitored during exposure in superfusion to NMDA, AMPA, epibatidine or high K⁺. NMDA -evoked [³H]NA release from EE hippocampal synaptosomes was significantly higher than that from SE synaptosomes, while the [³H]NA overflow elicited by 100 μM AMPA, 1 μM epibatidine or (9, 15, 25 mM) KCl was unchanged. In EE mice, the apparent affinity of NMDA or glycine was unmodified, while the efficacy was significantly augmented. Sensitivity to non-selective or subtype-selective NMDA receptor antagonists (MK-801, ifenprodil and Zn²⁺ ions) was not modified in EE. Finally, the analysis of NMDA receptor subunit mRNA expression in noradrenergic cell bodies of the locus coeruleus showed that NR1, NR2A, NR2B and NR2D subunits were unchanged, while NR2C decreased significantly in EE mice as compared to SE mice. Functional up-regulation of the pre-synaptic NMDA receptors modulating NA release might contribute to the improved learning and memory found in animals exposed to an EE.     

A Polyamine-Deficient Diet Prevents Oxaliplatin-Induced Acute Cold and Mechanical Hypersensitivity in Rats

Background

Oxaliplatin is an anticancer drug used for the treatment of advanced colorectal cancer, but it can also cause painful peripheral neuropathies. The pathophysiology of these neuropathies has not been yet fully elucidated, but may involve spinal N-methyl-D-aspartate (NMDA) receptors, particularly the NR2B subunit. As polyamines are positive modulators of NMDA-NR2B receptors and mainly originate from dietary intake, the modulation of polyamines intake could represent an interesting way to prevent/modulate neuropathic pain symptoms by opposing glutamate neurotransmission.

Methods

The effect of a polyamine deficient diet was investigated in an animal model of oxaliplatin-induced acute pain hypersensitivity using behavioral tests (mechanical and cold hypersensitivity). The involvement of spinal glutamate neurotransmission was monitored by using a proton nuclear magnetic resonance spectroscopy based metabolomic approach and by assessing the expression and phosphorylation of the NR2B subunit of the NMDA receptor.

Results

A 7-day polyamine deficient diet totally prevented oxaliplatin-induced acute cold hypersensitivity and mechanical allodynia. Oxaliplatin-induced pain hypersensitivity was not associated with an increase in NR2B subunit expression or phosphorylation, but with an increase of glutamate level in the spinal dorsal horn which was completely prevented by a polyamine deficient diet. As a validation that the oxaliplatin-induced hypersensitivity could be due to an increased activity of the spinal glutamate system, an intrathecal administration of the specific NR2B antagonist, ifenprodil, totally reversed oxaliplatin-induced mechanical and cold hypersensitivity.

Conclusion

A polyamine deficient diet could represent a promising and valuable nutritional therapy to prevent oxaliplatin-induced acute pain hypersensitivity.     

Increased expression of NR2A subunit does not alter NMDA-evoked responses in cultured fetal trisomy 16 mouse hippocampal neurons

The trisomy 16 (Ts16) mouse is an animal model for human trisomy 21 (Down's syndrome). The gene encoding the NR2A subunit of the NMDA receptor has been localized to mouse chromosome 16. In the present study, western blot analysis revealed a 2.5-fold increase of NR2A expression in cultured Ts16 embryonic hippocampal neurons. However, this increase did not affect the properties of NMDA-evoked currents in response to various modulators. The sensitivity of NMDA receptors to transient applications of NMDA, spermine, and Zn(2+) was investigated in murine Ts16 and control diploid cultured embryonic hippocampal neurons. Peak and steady-state currents evoked by NMDA were potentiated by spermine at concentrations < 1 mM, and inhibited by Zn(2+) in a dose-dependent and voltage-independent manner. No marked difference was observed between Ts16 and control diploid neurons for any of these modulators with regard to IC(50) and EC(50) values or voltage dependency. Additionally, inhibition by the NR2B selective inhibitor, ifenprodil, was similar. These results demonstrate that NMDA-evoked currents are not altered in cultured embryonic Ts16 neurons and suggest that Ts16 neurons contain similar functional properties of NMDA receptors as diploid control neurons despite an increased level of NR2A expression.     

Ethanol sensitivity of NMDA receptors

NMDA receptors are ionotropic glutamate receptors assembled of subunits of the NR1 and of the NR2 family (NR2A–NR2D). The subunit diversity largely affects the pharmacological properties of NMDA receptors and, hence, gives rise to receptor heterogeneity. As an overall result of studies on recombinant and native NMDA receptors, ethanol inhibits the function of receptors containing the subunits NR2A and/or NR2B to a greater extent than those containing NR2C or NR2D. For example, in rat cultured mesencephalic neurons, NR2C expression was developmentally increased, whereas expression of NR2A and NR2B was decreased. These changes coincided with a developmental loss of sensitivity of NMDA responses to ethanol and ifenprodil, a non-competitive NMDA receptor antagonist that shows selectivity for NR2B-containing receptors. Also in rat locus coeruleus neurons, the low ethanol sensitivity of somatic NMDA receptors could be explained by a prominent expression of NR2C. The inhibitory site of action for ethanol on the NMDA receptor is not yet known. Patch–clamp studies suggest a target site exposed to or only accessible from the extracellular environment. Apparently, amino acid residue Phe⁶³⁹, located in the TM3 domain of NR1, plays a crucial role in the inhibition of NMDA receptor function by ethanol. Since this phenylalanine site is common to all NMDA and non-NMDA receptor (AMPA/kainate receptor) subunits, this observation is consistent with accumulating evidence for a similar ethanol sensitivity of a variety of NMDA and non-NMDA receptors, but it cannot explain the differences in ethanol sensitivity observed with different NR2 subunits.     

Transcription of the NR1 subunit of the N-methyl-D-aspartate receptor is down-regulated by excitotoxic stimulation and cerebral ischemia

The N-methyl-D-aspartate (NMDA) type of glutamate receptor (NMDAR) plays central roles in normal and pathological neuronal functioning. We have examined the regulation of the NR1 subunit of the NMDAR in response to excessive activation of this receptor in in vitro and in vivo models of excitotoxicity. NR1 protein expression in cultured cortical neurons was specifically reduced by stimulation with 100 microM NMDA or glutamate. NMDA decreased NR1 protein amounts by 71% after 8 h. Low NMDA concentrations (< or = 10 microM) had no effect. NR1 down-regulation was inhibited by the general NMDAR antagonist DL-AP5 and also by ifenprodil, which specifically antagonizes NMDARs containing NR2B subunits. Arrest of NMDAR signaling with DL-AP5 after brief exposure to NMDA did not prevent subsequent NR1 decrease. Down-regulation of NR1 did not involve calpain cleavage but resulted from a decrease in de novo synthesis consequence of reduced mRNA amounts. In contrast, NMDA did not alter the expression of NR2A mRNA or newly synthesized protein. In neurons transiently transfected with an NR1 promoter/luciferase reporter construct, promoter activity was reduced by 68% after 2 h of stimulation with NMDA, and its inhibition required extracellular calcium. A similar mechanism of autoregulation of the receptor probably operates during cerebral ischemia, because NR1 mRNA and protein were strongly decreased at early stages of blood reperfusion in the infarcted brains of rats subjected to occlusion of the middle cerebral artery. Because NR1 is the obligatory subunit of NMDARs, this regulatory mechanism will be fundamental to NMDAR functioning.     

Reactive derivatives for affinity labeling in the ifenprodil site of NMDA receptors

To prepare thiol-reactive ifenprodil derivatives designed as potential probes for cysteine-substituted NR2B containing NMDA receptors, electrophilic centers were introduced in different areas of the ifenprodil structure. Intermediates and final compounds were evaluated by binding studies and by electrophysiology to determine the structural requirements for their selectivity. The reactive compounds were further tested for their stability and for their reactivity in model reactions; some were found suitable as structural probes to investigate the binding site and the docking mode of ifenprodil in the NR2B subunit.     

Identification and mechanism of action of two histidine residues underlying high-affinity Zn2+ inhibition of the NMDA receptor.

Zinc (Zn2+) inhibition of N-methyl-D-aspartate receptor (NMDAR) activity involves both voltage-independent and voltage-dependent components. Recombinant NR1/NR2A and NR1/NR2B receptors exhibit similar voltage-dependent block, but voltage-independent Zn2+ inhibition occurs with much higher affinity for NR1/NR2A than NR1/NR2B receptors (nanomolar versus micromolar IC50, respectively). Here, we show that two neighboring histidine residues on NR2A represent the critical determinant (termed the "short spacer") for high-affinity, voltage-independent Zn2+ inhibition using the Xenopus oocyte expression system and site-directed mutagenesis. Mutation of either one of these two histidine residues (H42 and H44) in the extracellular N-terminal domain of NR2A shifted the IC50 for high-affinity Zn2+ inhibition approximately 200-fold without affecting the EC50 of the coagonists NMDA and glycine. We suggest that the mechanism of high-affinity Zn2+ inhibition on the NMDAR involves enhancement of proton inhibition.