Regulation of polyamine and magnesium inhibition of the N-methyl-D-aspartate (NMDA) receptor ionophore complex.

The inhibitory effects of spermidine, putrescine and magnesium on [3H]TCP binding in crude synaptosomes and well washed buffy coat membranes were studied. We report that the IC50 values for the drugs tested varied depending on the state of activation of the channel, being lowest when the channel is maximally activated and highest when the channel is least activated in the control buffy coat preparation. Comparison of the inhibitory characteristics of these 3 agents when activated by glutamate, glycine, and low concentrations of spermidine or magnesium strongly suggest that putrescine and magnesium share a common inhibitory mechanism, perhaps mediated by the voltage-dependent channel site for magnesium. Inhibition by spermidine, however, is clearly mediated by a distinct site, non-identical to the phencyclidine receptor. Glutamate appears to uncouple this inhibition in about one-half of the N-methyl-D-aspartate (NMDA) complexes, suggesting the possibility of distinct subpopulations of NMDA-operated ion channels. The potential physiological relevance of these findings is discussed.     

NMDA受体在癫痫发病机制中的作用

癫痫是慢性反复发作短暂脑功能失调综合征,是神经科常见疾病之一。目前有关癫痫的发病机制尚未阐明。研究表明,癫痫的发生与兴奋性神经递质和抑制性神经递质的失衡有关。谷氨酸作为一种主要的兴奋性神经递质,通过受体介导的兴奋性机制在癫痫的发生过程中具有重要作用。谷氨酸受体可以分为促离子型和促代谢型2类。     

The N-methyl-D-aspartate (NMDA) receptor complex: a stoichiometric analysis of radioligand binding domains

A stoichiometric analysis of pharmacological domains within the N-methyl-D-aspartate (NMDA) receptor complex was made by evaluating the binding of L-[3H]glutamate, [3H]CPP, [3H]glycine and [3H]MK-801 to purified synaptic membranes isolated from rat telencephalon. The binding of all radioligands exhibited pharmacological and kinetic properties consistent with the labeling of homogeneous populations of sites associated with the NMDA receptor. However, strychnine-insensitive [3H]glycine binding sites were present at close to 2-fold the density of the other sites examined. These data, together with recent electrophysiological and receptor autoradiographic findings, are utilized as a basis for hypotheses regarding the ratio of transmitter recognition, allosteric and channel binding sites within the NMDA receptor complex.     

Ethanol and NMDA receptor signaling

NMDA receptors are a multi-subunit family of ionotropic receptors activated by the neurotransmitter glutamate. Localized primarily postsynaptically in neurons, they play an important role in mediating excitatory synaptic neurotransmission and are implicated in a wide variety of important calcium-dependent neuronal processes. Experimental animals expressing mutant forms of NMDA subunits display abnormal behaviors and locomotor and cognitive impairments. Over the last 10 years, a wealth of studies has indicated that NMDA receptors are an important site of action for ethanol in the brain. The effects of acute ethanol on NMDA receptor function is discussed herein, with particular focus on efforts to define a molecular site of action of ethanol on the receptor. While it is clear that the ethanol sensitivity of NMDA receptors is influenced by subunit composition, it is also apparent that posttranslational factors such as phosphorylation and protein-protein interactions are important in modulating this sensitivity.     

Dynamic regulation of NMDA receptor transmission

N-methyl-D-aspartate receptors (NMDARs) are critical for establishing, maintaining, and modifying glutamatergic synapses in an activity-dependent manner. The subunit composition, synaptic expression, and some of the properties of NMDARs are regulated by synaptic activity, affecting processes like synaptic plasticity. NMDAR transmission is dynamic, and we were interested in studying the effect of acute low or null synaptic activity on NMDA receptors and its implications for synaptic plasticity. Periods of no stimulation or low-frequency stimulation increased NMDAR transmission. Changes became stable after periods of 20 min of low or no stimulation. These changes in transmission have a postsynaptic origin and are explained by incorporation of GluN2B-containing receptors to synapses. Importantly, periods of low or no stimulation facilitate long-term potentiation induction. Moreover, recovery after a weak preconditioning stimulus that normally blocks subsequent potentiation is facilitated by a nonstimulation period. Thus synaptic activity dynamically regulates the level of NMDAR transmission adapting constantly the threshold for plasticity.     

Molecular determinants of NMDA receptor internalization

Although synaptic AMPA receptors have been shown to rapidly internalize, synaptic NMDA receptors are reported to be static. It is not certain whether NMDA receptor stability at synaptic sites is an inherent property of the receptor, or is due to stabilization by scaffolding proteins. In this study, we demonstrate that NMDA receptors are internalized in both heterologous cells and neurons, and we define an internalization motif, YEKL, on the distal C-terminus of NR2B. In addition, we show that the synaptic protein PSD-95 inhibits NR2B-mediated internalization, and that deletion of the PDZ-binding domain of NR2B increases internalization in neurons. This suggests an involvement for PSD-95 in NMDA receptor regulation and an explanation for NMDA receptor stability at synaptic sites.     

Dantrolene antagonizes the glycineB site of the NMDA receptor

In this study we tested the hypothesis that dantrolene, an established inhibitor of the skeletal muscle isoform of the ryanodine receptor, may interfere with activity of NMDA receptors in neurons. We assessed the effects of dantrolene on [(3)H]MK-801 and [(3)H]glycine binding to isolated rat cortical membranes. Dantrolene inhibited [(3)H]MK-801 binding in the presence of 100 microM NMDA with an IC(50) of 58.4 microM. The IC(50) value increased to 99.6, 343.0 and 364.6 microM in the presence of 10, 30 and 50 microM glycine, respectively, suggesting that dantrolene competes with glycine for binding site at the NMDA receptor complex. A binding assay using [(3)H]glycine confirmed this supposition: dantrolene inhibited strychnine-insensitive glycine binding in a dose-dependent way. Thus, our results show that dantrolene at concentrations of 50-100 microM and higher blocks the glycine binding site of the NMDA receptor complex and in this way inhibits activation of the NMDA ion channel. These data reveal a new mechanism of dantrolene action in neuronal tissue. Our results also suggest that the neuroprotective effect of dantrolene may be at least partly explained by its activity as a non-competitive antagonist of NMDA receptors.     

NMDA receptor kinetics and synaptic function

Presynaptic release of L-glutamate mediates neurotransmission at most excitatory synapses in the vertebrate central nervous system. At the postsynaptic membrane, glutamate binds to two classes of ligand-gated ion channels, AMPA receptors and NMDA receptors. These channels are the basis of the two kinetically distinct components of the excitatory postsynaptic current (epsc). The slower synaptic conductance is mediated by NMDA receptor channels which, after binding glutamate, activate slowly and can remain activated for several hundred milliseconds. The average latency between glutamate binding and channel opening is at least several milliseconds and may be much longer. If the time to first opening is short many fewer channels would be required at each synaptic site to account for the amplitude of the NMDA receptor component of spontaneous miniature epscs, than if the time to first opening is very long.     

NMDA receptor delayed maturation and schizophrenia

This paper presents the hypothesis that NMDA receptor delayed maturation (NRDM) may lead to the pathogenesis of schizophrenic psychotic symptoms. This hypothesis is further analyzed in the language of a neural modeling formulation. This formulation points to a possible chain of pathological events, leading from molecular-level NRDM to over-increased synaptic plasticity, and to the formation of pathological attractors, a putative macroscopic-level correlate of schizophrenic positive symptoms. The relations of the NRDM hypothesis to other alterations which are assumed to take place in schizophrenia are discussed, together with possible ways to test this hypothesis.     

Regulation of NMDA receptor trafficking by amyloid-beta

Amyloid-beta peptide is elevated in the brains of patients with Alzheimer disease and is believed to be causative in the disease process. Amyloid-beta reduces glutamatergic transmission and inhibits synaptic plasticity, although the underlying mechanisms are unknown. We found that application of amyloid-beta promoted endocytosis of NMDA receptors in cortical neurons. In addition, neurons from a genetic mouse model of Alzheimer disease expressed reduced amounts of surface NMDA receptors. Reducing amyloid-beta by treating neurons with a gamma-secretase inhibitor restored surface expression of NMDA receptors. Consistent with these data, amyloid-beta application produced a rapid and persistent depression of NMDA-evoked currents in cortical neurons. Amyloid-beta-dependent endocytosis of NMDA receptors required the alpha-7 nicotinic receptor, protein phosphatase 2B (PP2B) and the tyrosine phosphatase STEP. Dephosphorylation of the NMDA receptor subunit NR2B at Tyr1472 correlated with receptor endocytosis. These data indicate a new mechanism by which amyloid-beta can cause synaptic dysfunction and contribute to Alzheimer disease pathology.     

PET studies of [18F]methyl-MK-801, a potential NMDA receptor complex radioligand.

Using positron emission tomography (PET), the potential of 18F-labelled fluoro-methyl-MK-801([18F]FMM) as a radioligand for in vivo studies of the NMDA receptor complex was investigated in baboons. In baseline conditions, there was a slight differential retention of [18F]FMM in cerebral cortex and striatum relative to cerebellum, compatible with specific binding. However, neither pretreatment with pharmacological doses of MK-801 or phencyclidine, nor severe, transient brain hypoxia, were able to clearly alter [18]FMM brain regional kinetics, indicating limited usefulness of this radioligand for in vivo PET investigations of the NMDA receptor.     

Single channel properties of the N-methyl-D-aspartate receptor channel using NMDA and NMDA agonists: on-cell recordings

Summary The cell-attached patch-clamp mode has been applied in cultured rat hippocampal neurons to record single channel currents through the ion channel which is coupled to activation of the N-methyl-D-aspartate (NMDA) receptor. A channel, with a conductance of 37 pS, was studied with either NMDA or D-cis-1-amino-1,3-cyclopentanedicarboxylic acid (ACPD) in the patch pipette. The mean open time of the channels with NMDA in the pipette was near 5 ms at -80 mV and was diminished about 0.7 ms for each 20 mV of hyperpolarization. The mean open times with ACPD in the pipette were longer at all voltages studied and for both agonists the mean open times showed an exponential dependence on patch potential. Distributions for the channel open times were generally well-fit with single exponentials, whereas distributions for closed times required two-component fits. In some instances, the open distributions also showed a second, rapid time component. When Mg² was included in the pipette (40 or 100 M), the mean open times were significantly diminished with the effect increasing with the magnitude of the hyperpolarization. Both the amplitudes and mean open times of the NMDA channel were strongly modulated by temperature with Q₁₀ values in excess of 2.5.     

NMDA receptor dependent paroxysmal discharges in the dentate gyrus

The N-methyl-D-aspartic acid subclass of glutamate receptors is thought to be involved in events that depend on repetitive activation of neurons. The present study shows that repetitive stimulation of CA3 can produce epileptiform discharges in the contralateral dentate gyrus. This distinctive paroxysmal response was blocked by the NMDA antagonists, ketamine and MK-801. The MK-801 blockade required previous activation of the dentate gyrus in the presence of the drug, thus demonstrating, in vivo, a use-dependent effect. These studies show that repetitive, low intensity stimulation of hippocampal circuits can produce NMDA-mediated epileptiform discharges in the dentate gyrus.     

NMDA receptor antagonists extend the sensitive period for imprinting

Filial imprinting in the domestic chick occurs during a sensitive period of development. The exact timing of this period can vary according to the methods used to measure imprinting. Using our imprinting paradigm, we have shown that normal, dark-reared chicks lose the ability to imprint after the second day post-hatching. Further, we reported that chicks treated 10 h after hatching with a mixture of the noncompetitive NMDA receptor antagonist ketamine (55 mg/kg) and the alpha(2)-adrenergic receptor agonist xylazine (6 mg/kg) were able to imprint on day 8 after hatching, whereas controls treated with saline did not imprint. We now show that the effect of the ketamine-xylazine mixture can be mimicked by treating chicks with ketamine alone or with another noncompetitive NMDA receptor antagonist, MK-801 (5 mg/kg). Treating chicks with a single dose of ketamine (55 mg/kg) or with a single dose of xylazine (6 mg/kg) failed to produce the effect on the sensitive period. However, prolonging the action of ketamine by treating chicks with two doses of ketamine (at 10 and 12 h after hatching) did allow imprinting on day 8. In contrast, prolonging the action of xylazine had no effect on the sensitive period for imprinting. Chicks treated with MK-801 were also able to imprint on day 8. Thus, we have evidence that the NMDA receptor system is involved in the mechanisms that control the sensitive period for imprinting.     

NMDA receptor permeation: a light in the tunnel

A single amino acid residue determines single-channel conductance, ion permeation and channel block in the NMDA receptor, three of the key features of this ligand-gated ion channel.     

Subunit- and site-specific pharmacology of the NMDA receptor channel.

N-Methyl-D-aspartate (NMDA) receptor channels play important roles in various physiological functions such as synaptic plasticity and synapse formation underlying memory, learning and formation of neural networks during development. They are also important for a variety of pathological states including acute and chronic neurological disorders, psychiatric disorders, and neuropathic pain syndromes. cDNA cloning has revealed the molecular diversity of NMDA receptor channels. The identification of multiple subunits with distinct distributions, properties and regulation, implies that NMDA receptor channels are heterogeneous in their pharmacological properties, depending on the brain region and the developmental stage. Furthermore, mutation studies have revealed a critical role for specific amino acid residues in certain subunits in determining the pharmacological properties of NMDA receptor channels. The molecular heterogeneity of NMDA receptor channels as well as their dual role in physiological and pathological functions makes it necessary to develop subunit- and site-specific drugs for precise and selective therapeutic intervention. This review summarizes from a molecular perspective the recent advances in our understanding of the pharmacological properties of NMDA receptor channels with specific references to agonists binding sites, channel pore regions, allosteric modulation sites for protons, polyamines, redox agents, Zn2+ and protein kinases, phosphatases.