Polymodal regulation of NMDA receptor channels

Glutamate-activated N-methyl-D-aspartate (NMDA) receptors are ligand-gated ion channels, which mediate synaptic transmission, long-term potentiation, synaptic plasticity and neurodegeneration via conditional Ca(2+) signalling. Recent crystallographic studies have focussed on solving the structural determinant of the ligand binding within the core region of NR1 and NR2 subunits. Future structural analysis will help to understand the mechanism of native channel activation and regulation during synaptic transmission. A number of NMDA receptor ligands have been identified which act as positive or negative modulators of receptor function. There is evidence that the lipid bilayer can further regulate the activity of the NMDA receptor channels. Modulators of NMDA receptor function offer the potential for the development of novel therapeutics to target neurological disorders associated with this family of glutamate ion channel receptors. Here, we review the recent literature concerning structural and functional properties, as well as the physiological and pathological roles of NMDA receptor channels.     

Subunit assembly of N-methyl-d-aspartate receptors analyzed by fluorescence resonance energy transfer

N-methyl-d-aspartate (NMDA) receptors play major roles in synaptic transmission and plasticity, as well as excitotoxicity. NMDA receptors are thought to be tetrameric complexes mainly composed of NMDA receptor (NR)1 and NR2 subunits. The NR1 subunits are required for the formation of functional NMDA receptor channels, whereas the NR2 subunits modify channel properties. Biochemical and functional studies indicate that subunits making up NMDA receptors are organized into a dimer of dimers, and the N termini of the subunits are major determinants for receptor assembling. Here we used a biophysical approach, fluorescence resonance energy transfer, to analyze the assembly of intact, functional NMDA receptors in living cells. The results showed that NR1, NR2A, and NR2B subunits could form homodimers when they were expressed alone in HEK293 cells. Subunit homodimers were also found existing in heteromeric NMDA receptors formed between NR1 and NR2 subunits. These findings are consistent with functional NMDA receptors being arranged as a dimer of dimers. In addition, our data indicated that the conformation of NR1 subunit homodimers was affected by the partner NR2 subunits during the formation of heteromeric receptor complexes, which might underlie the mechanism by which NR2 subunits modify NMDA receptor function.     

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.     

Different structural requirements for functional ion pore transplantation suggest different gating mechanisms of NMDA and kainate receptors

Although considerable progress has been made in characterizing the physiological function of the high-affinity kainate (KA) receptor subunits KA1 and KA2, no homomeric ion channel function has been shown. An ion channel transplantation approach was employed in this study to directly test if homomerically expressed KA1 and KA2 pore domains are capable of conducting currents. Transplantation of the ion pore of KA1 or KA2 into GluR6 generated perfectly functional ion channels that allowed characterization of those electrophysiological and pharmacological properties that are determined exclusively by the ion pore of KA1 or KA2. This demonstrates for the first time that KA1 and KA2 ion pore domains are intrinsically capable of conducting ions even in homomeric pore assemblies. NMDA receptors, similar to KA1- or KA2-containing receptors, function only as heteromeric complexes. They are composed of NR1 and NR2 subunits, which both are non-functional when expressed homomerically. In contrast to NR1, the homomeric NR2B ion pore failed to translate ligand binding into pore opening when transplanted into GluR6. Similarly, heteromeric coexpression of the ion channel domains of both NR1 and NR2 inserted into GluR6 failed to produce functional channels. Therefore, we conclude that the mechanism underlying the ion channel opening in the obligatorily heterotetrameric NMDA receptors differs significantly from that in the facultatively heterotetrameric alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate and KA receptors.     

Principal role of NR3 subunits in NR1/NR3 excitatory glycine receptor function

Calcium-permeable N-methyl-d-aspartate (NMDA) receptors are tetrameric cation channels composed of glycine-binding NR1 and glutamate-binding NR2 subunits, which require binding of both glutamate and glycine for efficient channel gating. In contrast, receptors assembled from NR1 and NR3 subunits function as calcium-impermeable excitatory glycine receptors that respond to agonist application only with low efficacy. Here, we show that antagonists of and substitutions within the glycine-binding site of NR1 potentiate NR1/NR3 receptor function up to 25-fold, but inhibition or mutation of the NR3 glycine binding site reduces or abolishes receptor activation. Thus, glycine bound to the NR1 subunit causes auto-inhibition of NR1/NR3 receptors whereas glycine binding to the NR3 subunits is required for opening of the ion channel. Our results establish differential roles of the high-affinity NR3 and low-affinity NR1 glycine-binding sites in excitatory glycine receptor function.     

Biochemical studies of the structure and function of theN-methyl-D-aspartate subtype of glutamate receptors

TheN-methyl-D-aspartate (NMDA) subtype of glutamate receptors plays a key role in synaptic transmission, synaptic plasticity, synaptogenesis, and excitotocity in the mammalian central nervous system. The NMDA receptor channel is formed from two gene products from two glutamate receptor subunit families, termed NR1 and NR2. Although the subunit composition of native NMDA receptors is incompletely understood, electrophysiological studies using recombinant receptors suggest that functional NMDA receptors consist of heteromers containing combinations of NR1, which is essential for channel activity, and NR2, which modulates the properties of the channels. The lack of agonists or antagonists selective for a given subunit of NMDA receptors has made it difficult to understand the subunit expression, subunit composition, and posttranslational modification mechanisms of native NMDA receptors. Therefore, most studies on NMDA receptors that examine regional expression and ontogeny have been focused at the level of the mRNAs encoding the different subunits using northern blotting, ribonuclease protection, andin situ hybridization techniques. However, the data from these studies do not provide clear information about the resultant subunit protein. To directly examine the protein product of the NMDA receptor subunit genes, the development of subunit-specific antibodies using peptides and fusion proteins has provided a good approach for localizing, quantifying, and characterizing the receptor subunits in tissues and transfected cell lines, and to study the subunit composition and the functional effects of posttranslational processing of the NMDA subunits, particularly the phosphorylation profiles of NMDA glutamate receptors.     

Conserved structural and functional control of N-methyl-D-aspartate receptor gating by transmembrane domain M3

The molecular events controlling glutamate receptor ion channel gating are complex. The movement of transmembrane domain M3 within N-methyl-d-aspartate (NMDA) receptor subunits has been suggested to be one structural determinant linking agonist binding to channel gating. Here we report that covalent modification of NR1-A652C or the analogous mutation in NR2A, -2B, -2C, or -2D by methanethiosulfonate ethylammonium (MT-SEA) occurs only in the presence of glutamate and glycine, and that modification potentiates recombinant NMDA receptor currents. The modified channels remain open even after removing glutamate and glycine from the external solution. The degree of potentiation depends on the identity of the NR2 subunit (NR2A < NR2B < NR2C,D) inversely correlating with previous measurements of channel open probability. MTSEA-induced modification of channels is associated with increased glutamate potency, increased mean single-channel open time, and slightly decreased channel conductance. Modified channels are insensitive to the competitive antagonists D-2-amino-5-phosphonovaleric acid (APV) and 7-Cl-kynurenic acid, as well as allosteric modulators of gating (extracellular protons and Zn(2+)). However, channels remain fully sensitive to Mg(2+) blockade and partially sensitive to pore block by (+)MK-801, (-)MK-801, ketamine, memantine, amantadine, and dextrorphan. The partial sensitivity to (+)MK-801 may reflect its ability to stimulate agonist unbinding from MT-SEA-modified receptors. In summary, these data suggest that the SYTANLAAF motif within M3 is a conserved and critical determinant of channel gating in all NMDA receptors.     

Ethanol inhibition of NMDA receptors under conditions of altered protein kinase A activity

N-methyl-D-aspartate receptors (NMDA) are glutamate-activated ligand-gated ion channels that participate in diverse forms of synaptic plasticity as well as glutamate-dependent excitotoxicity. Inhibition of the NMDA receptor function may underlie some of the behavioral actions associated with acute exposure to ethanol. The sensitivity of NMDA receptors to ethanol is influenced by the subunit composition of the receptor and, by association, with certain cytoskeletal proteins. Previous studies have also suggested that phosphorylation may regulate the sensitivity of NMDA receptors to ethanol. In this study, the ethanol inhibition of recombinant NMDA receptor currents was determined under conditions designed to enhance or inhibit the activity of protein kinase A (PKA). Human embryonic kidney 293 (HEK293) cells were transfected with cDNAs encoding NMDA subunits and channel activity was monitored with whole-cell patch-clamp electrophysiology. Under control recording conditions, ethanol (100 mM) inhibited NR1/2A and NR1/2B receptor currents by approximately 25-30%. The degree of ethanol inhibition was not affected or was slightly enhanced under conditions designed to enhance PKA activity. This included treatment of cells with cAMP analogs, inclusion of phosphatase inhibitors or purified PKA in the pipette filling solution, co-expression of catalytically active PKA, expression of the NR1 PKA-site phosphorylation site mimic (S897D) or by co-expression of the PKA scaffolding protein yotiao or the dopamine D(1) receptor. Ethanol inhibition of NR1/2A and NR1/2B receptors was not altered when PKA effects were suppressed, either by co-expression of a PKI inhibitory peptide or the phosphorylation-deficient NR1 mutants (S897A, S896A, S896A/S897A). In addition, ethanol inhibition of NMDA-induced currents in cultured cortical or hippocampal neurons was not affected by modulators of PKA. These results suggest that PKA does not appear to play a major role in determining the acute ethanol sensitivity of NMDA receptors.     

Subcellular segregation of distinct heteromeric NMDA glutamate receptors in the striatum

Functional N-methyl-d-aspartate (NMDA) glutamate receptors are composed of heteromeric complexes of NR1, the obligatory subunit for channel activity, and NR2 or NR3 family members, which confer variability in the properties of the receptors. Recent studies have provided evidence for the existence of both binary (containing NR1 and either NR2A or NR2B) and ternary (containing NR1, NR2A, and NR2B) receptor complexes in the adult mammalian brain. However, the mechanisms regulating subunit assembly and receptor localization are not well understood. In the CNS, NMDA subunits are present both at intracellular sites and the post-synaptic membrane of neurons. Using biochemical protein fractionation and co-immunoprecipitation approaches we have found that in rat striatum binary NMDA receptors are widely distributed, and can be identified in the light membrane, synaptosomal membrane, and synaptic vesicle-enriched subcellular compartments. In contrast, ternary receptors are found exclusively in the synaptosomal membranes. When striatal proteins are chemically cross-linked prior to subcellular fractionation, ternary NMDA receptors can be precipitated from the light membrane and synaptic vesicle-enriched fractions where this type of receptor complex is not detectable under normal conditions. These findings suggest differential targeting of distinct types of NMDA receptor assemblies between intracellular and post-synaptic sites based on subunit composition. This targeting may underlie important differences in the regulation of the transport pathways involved in both normal as well as pathological receptor functions.     

Structure of the zinc‐bound amino‐terminal domain of the NMDA receptor NR2B subunit

N‐methyl‐D ‐aspartate (NMDA) receptors belong to the family of ionotropic glutamate receptors (iGluRs) that mediate the majority of fast excitatory synaptic transmission in the mammalian brain. One of the hallmarks for the function of NMDA receptors is that their ion channel activity is allosterically regulated by binding of modulator compounds to the extracellular amino‐terminal domain (ATD) distinct from the L ‐glutamate‐binding domain. The molecular basis for the ATD‐mediated allosteric regulation has been enigmatic because of a complete lack of structural information on NMDA receptor ATDs. Here, we report the crystal structures of ATD from the NR2B NMDA receptor subunit in the zinc‐free and zinc‐bound states. The structures reveal the overall clamshell‐like architecture distinct from the non‐NMDA receptor ATDs and molecular determinants for the zinc‐binding site, ion‐binding sites, and the architecture of the putative phenylethanolamine‐binding site.     

N-甲基-D-天氡氨酸受体的分子结构与生理功能

NMDA(N-甲基-D-天氡氨酸)受体是离子型谷氨酸受体的一种亚型,在中枢神经系统的突触传递和突触可塑性调节中起着重要的作用。生物体内已经发现了三种NMDA受体亚基,通过基因的选择性剪切可产生多种亚单位。NMDA受体是一个具有多个结合位点的大分子复合物,其生理特性同异聚体通道的装配密切相关。NMDA受体的异常会导致一些认知功能的缺失,这为治疗性药物开发提供了靶点。     

Subtype-specific enhancement of NMDA receptor currents by mutant huntingtin

Evidence suggests that NMDA receptor-mediated neurotoxicity plays a role in the selective neurodegeneration underlying Huntington's disease (HD). The gene mutation that causes HD encodes an expanded polyglutamine tract of >35 in huntingtin, a protein of unknown function. Both huntingtin and NMDA receptors interact with cytoskeletal proteins, and, for NMDA receptors, such interactions regulate surface expression and channel activity. To determine whether mutant huntingtin alters NMDA receptor expression or function, we coexpressed mutant or normal huntingtin, containing 138 or 15 glutamine repeats, respectively, with NMDA receptors in a cell line and then assessed receptor channel function by patch-clamp recording and surface expression by western blot analysis. It is interesting that receptors composed of NR1 and NR2B subunits exhibited significantly larger currents when coexpressed with mutant compared with normal huntingtin. Moreover, this effect was selective for NR1/NR2B, as NR1/NR2A showed similar currents when coexpressed with mutant versus normal huntingtin. However, ion channel properties and total surface expression of the NR1 subunit were unchanged in cells cotransfected with NR1/NR2B and mutant huntingtin. Our results suggest that mutant huntingtin may increase numbers of functional NR1/NR2B-type receptors at the cell surface. Because NR1/NR2B is the predominant NMDA receptor subtype expressed in medium spiny neostriatal neurons, our findings may help explain the selective vulnerability of these neurons in HD.     

Molecular mechanism of ligand recognition by NR3 subtype glutamate receptors

NR3 subtype glutamate receptors have a unique developmental expression profile, but are the least well-characterized members of the NMDA receptor gene family, which have key roles in synaptic plasticity and brain development. Using ligand binding assays, crystallographic analysis, and all atom MD simulations, we investigate mechanisms underlying the binding by NR3A and NR3B of glycine and D-serine, which are candidate neurotransmitters for NMDA receptors containing NR3 subunits. The ligand binding domains of both NR3 subunits adopt a similar extent of domain closure as found in the corresponding NR1 complexes, but have a unique loop 1 structure distinct from that in all other glutamate receptor ion channels. Within their ligand binding pockets, NR3A and NR3B have strikingly different hydrogen bonding networks and solvent structures from those found in NR1, and fail to undergo a conformational rearrangement observed in NR1 upon binding the partial agonist ACPC. MD simulations revealed numerous interdomain contacts, which stabilize the agonist-bound closed-cleft conformation, and a novel twisting motion for the loop 1 helix that is unique in NR3 subunits.     

The effects of NR2 subunit-dependent NMDA receptor kinetics on synaptic transmission and CaMKII activation

N-Methyl-D-aspartic acid (NMDA) receptors are widely expressed in the brain and are critical for many forms of synaptic plasticity. Subtypes of the NMDA receptor NR2 subunit are differentially expressed during development; in the forebrain, the NR2B receptor is dominant early in development, and later both NR2A and NR2B are expressed. In heterologous expression systems, NR2A-containing receptors open more reliably and show much faster opening and closing kinetics than do NR2B-containing receptors. However, conflicting data, showing similar open probabilities, exist for receptors expressed in neurons. Similarly, studies of synaptic plasticity have produced divergent results, with some showing that only NR2A-containing receptors can drive long-term potentiation and others showing that either subtype is capable of driving potentiation. In order to address these conflicting results as well as open questions about the number and location of functional receptors in the synapse, we constructed a Monte Carlo model of glutamate release, diffusion, and binding to NMDA receptors and of receptor opening and closing as well as a model of the activation of calcium-calmodulin kinase II, an enzyme critical for induction of synaptic plasticity, by NMDA receptor-mediated calcium influx. Our results suggest that the conflicting data concerning receptor open probabilities can be resolved, with NR2A- and NR2B-containing receptors having very different opening probabilities. They also support the conclusion that receptors containing either subtype can drive long-term potentiation. We also are able to estimate the number of functional receptors at a synapse from experimental data. Finally, in our models, the opening of NR2B-containing receptors is highly dependent on the location of the receptor relative to the site of glutamate release whereas the opening of NR2A-containing receptors is not. These results help to clarify the previous findings and suggest future experiments to address open questions concerning NMDA receptor function.     

H-Ras modulates N-methyl-D-aspartate receptor function via inhibition of Src tyrosine kinase activity

Tyrosine phosphorylation of the NR2A and NR2B subunits of the N-methyl-d-aspartate (NMDA) receptor by Src protein-tyrosine kinases modulates receptor channel activity and is necessary for the induction of long term potentiation (LTP). Deletion of H-Ras increases both NR2 tyrosine phosphorylation and NMDA receptor-mediated hippocampal LTP. Here we investigated whether H-Ras regulates phosphorylation and function of the NMDA receptor via Src family protein-tyrosine kinases. We identified Src as a novel H-Ras binding partner. H-Ras bound to Src but not Fyn both in vitro and in brain via the Src kinase domain. Cotransfection of H-Ras and Src inhibited Src activity and decreased NR2A tyrosine phosphorylation. Treatment of rat brain slices with Tat-H-Ras depleted NR2A from the synaptic membrane, decreased endogenous Src activity and NR2A phosphorylation, and decreased the magnitude of hippocampal LTP. No change was observed for NR2B. We suggest that H-Ras negatively regulates Src phosphorylation of NR2A and retention of NR2A into the synaptic membrane leading to inhibition of NMDA receptor function. This mechanism is specific for Src and NR2A and has implications for studies in which regulation of NMDA receptor-mediated LTP is important, such as synaptic plasticity, learning, and memory and addiction.     

AMPA receptor-dependent clustering of synaptic NMDA receptors is mediated by Stargazin and NR2A/B in spinal neurons and hippocampal interneurons

Under standard conditions, cultured ventral spinal neurons cluster AMPA- but not NMDA-type glutamate receptors at excitatory synapses on their dendritic shafts in spite of abundant expression of the ubiquitous NMDA receptor subunit NR1. We demonstrate here that the NMDA receptor subunits NR2A and NR2B are not routinely expressed in cultured spinal neurons and that transfection with NR2A or NR2B reconstitutes the synaptic targeting of NMDA receptors and confers on exogenous application of the immediate early gene product Narp the ability to cluster both AMPA and NMDA receptors. The use of dominant-negative mutants of GluR2 further showed that the synaptic targeting of NMDA receptors is dependent on the presence of synaptic AMPA receptors and that synaptic AMPA and NMDA receptors are linked by Stargazin and a MAGUK protein. This system of AMPA receptor-dependent synaptic NMDA receptor localization was preserved in hippocampal interneurons but reversed in hippocampal pyramidal neurons.     

Inducible expression and pharmacological characterization of recombinant rat NR1a/NR2A NMDA receptors

In this study, we have established a non-neuronal cell line stably and inducibly expressing recombinant NMDA receptors (NRs) composed of rat NR1a/NR2A subunits. EcR-293 cells were transfected with rat NR1a and NR2A cDNAs using the inducible mammalian expression vector pIND. Cell colonies resistant for the selecting agents were picked and tested for NR2A mRNA as well as protein expression using quantitative RT-PCR and flow cytometry based immunocytochemistry. Clonal cells expressing functional NMDA receptors were identified by measuring NMDA-evoked ion currents, and NMDA-induced increase in cytosolic free calcium concentration in whole-cell patch-clamp and fluorimetric calcium measurements, respectively. One clone named D5/H3, which exhibited the highest response to NMDA, was chosen to examine inducibility of the expression and for pharmacological profiling of recombinant NR1a/NR2A NMDA receptors. To check inducibility, NR2A subunit expression in D5/H3 cells treated with the inducing agent muristerone A (MuA) was compared with that in non-induced cells. Both NR2A mRNA and protein expression was several folds higher in cells treated with the inducing agent. As part of the pharmacological characterization, we examined the activation of the expressed NR1a/NR2A receptors as a function of increasing concentration of NMDA. NMDA-evoked concentration-dependent increases in cytosolic [Ca2+] with an EC50 value of 41 +/- 1 microM. In addition, whereas the NMDA response was concentration-dependently inhibited by the channel blocker MK-801 (IC50 = 58 +/- 6 nM), NR2B subunit selective NMDA receptor antagonists were ineffective. Thus, this cell line, which stably and inducibly expresses recombinant NR1a/NR2A NMDA receptors, can be a useful tool for testing NMDA receptor antagonists and studying their subunit selectivity.     

Fyn-mediated phosphorylation of NR2B Tyr-1336 controls calpain-mediated NR2B cleavage in neurons and heterologous systems

Cleavage of the intracellular carboxyl terminus of the N-methyl-d-aspartate (NMDA) receptor 2 subunit (NR2) by calpain regulates NMDA receptor function and localization. Here, we show that Fyn-mediated phosphorylation of NR2B controls calpain-mediated NR2B cleavage. In cultured neurons, calpain-mediated NR2B cleavage is significantly attenuated by blocking NR2B phosphorylation of Tyr-1336, but not Tyr-1472, via inhibition of Src family kinase activity or decreasing Fyn levels by small interfering RNA. In HEK cells, mutation of Tyr-1336 eliminates the potentiating effect of Fyn on calpain-mediated NR2B cleavage. The potentiation of NR2B cleavage by Fyn is limited to cell surface receptors and is associated with calpain translocation to plasma membranes during NMDA receptor activation. Finally, reducing full-length NR2B by calpain does not decrease extrasynaptic NMDA receptor function, and truncated NR1/2B receptors similar to those generated by calpain have electrophysiological properties matching those of wild-type receptors. Thus, the Fyn-controlled regulation of NMDA receptor cleavage by calpain may play critical roles in controlling NMDA receptor properties during synaptic plasticity and excitotoxicity.     

Activity-dependent shedding of the NMDA receptor glycine binding site by matrix metalloproteinase 3: a PUTATIVE mechanism of postsynaptic plasticity

Functional and structural alterations of clustered postsynaptic ligand gated ion channels in neuronal cells are thought to contribute to synaptic plasticity and memory formation in the human brain. Here, we describe a novel molecular mechanism for structural alterations of NR1 subunits of the NMDA receptor. In cultured rat spinal cord neurons, chronic NMDA receptor stimulation induces disappearance of extracellular epitopes of NMDA receptor NR1 subunits, which was prevented by inhibiting matrix metalloproteinases (MMPs). Immunoblotting revealed the digestion of solubilized NR1 subunits by MMP-3 and identified a fragment of about 60 kDa as MMPs-activity-dependent cleavage product of the NR1 subunit in cultured neurons. The expression of MMP-3 in the spinal cord culture was shown by immunoblotting and immunofluorescence microscopy. Recombinant NR1 glycine binding protein was used to identify MMP-3 cleavage sites within the extracellular S1 and S2-domains. N-terminal sequencing and site-directed mutagenesis revealed S542 and L790 as two putative major MMP-3 cleavage sites of the NR1 subunit. In conclusion, our data indicate that MMPs, and in particular MMP-3, are involved in the activity dependent alteration of NMDA receptor structure at postsynaptic membrane specializations in the CNS.     

Calcium influx through NMDA receptors, chronic receptor inhibition by ethanol and 2-amino-5-phosponopentanoic acid, and receptor protein expression

Chronic treatment of neurons with either ethanol or competitive and noncompetitive antagonists of NMDA receptors leads to enhanced expression of NMDA receptor density and function in neurons. The signal transduction pathways for such receptor up-regulation are not known. The focus of the present study was on the role of Ca2+ entry into neurons, either through receptor or voltage-gated channels, in the expression of the NMDA receptor subunit NR1 and the 71-kDa glutamate-binding protein (GBP) of a glutamate/NMDA receptor-like complex. Chronic inhibition of NMDA receptors in cortical neurons in primary cultures by either 100 mM ethanol or 100 microM 2-amino-5-phosphonopentanoic acid (2-AP5) increased the expression of NR1 and GBP. The effect of 2-AP5 on the expression of the two proteins was not additive with that of ethanol when neuronal cultures were treated with both agents at the same time. However, the effects of ethanol on NR1 and GBP expression were blocked by the simultaneous treatment with NMDA (50 microM). Activation or inhibition of other glutamate ionotropic receptors had no effect on the expression of NR1 and GBP. The inhibition of L- or N-type voltage-sensitive Ca2+ channels and voltage-gated Na+ channels also had little effect on the expression of either protein; neither did exposure of neurons to elevated extracellular Ca2+ concentrations (3 or 5 mM). On the other hand, treatment of neurons for 48 h with the intracellular Ca2+ chelator BAPTA-AM as well as partial chelation of extracellular Ca2+ with EGTA caused an up-regulation in NR1 and GBP expression. The enhanced expression of NR1 in neurons treated for 48 h with either ethanol or EGTA was correlated with increases in the activity of NMDA receptors demonstrated as a doubling of the NMDA-stimulated rise in intracellular free Ca2+ concentration. The effects of chronic administration of EGTA on both NR1 expression as well as NMDA receptor function were probably related to an acute inhibition by EGTA of NMDA-induced Ca2+ influx into neurons. It appears that the expression of both the NR1 subunit of NMDA receptors and the GBP of a receptor-like complex is regulated by intracellular Ca2+, especially that entering through NMDA receptor ion channels.