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.     

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.     

Ethanol Inhibition of Recombinant Heteromeric NMDA Channels in the Presence and Absence of Modulators

Abstract: The NMDA receptor/channel has been shown to be inhibited by ethanol in the clinically relevant range 25–100 m M . We studied heteromeric assemblies (NR1b/NR2) of the NMDA receptor, expressed in oocytes, to address three questions regarding this inhibition, and discovered the following: (1) The inhibition was nearly equivalent when ethanol was coapplied with the agonist, and when ethanol was introduced after steady-state current was established, suggesting that ethanol does not act by interfering with the activation process of the NMDA receptor. (2) The degree of inhibition was controlled by the NR2 subunit, with both NR2A and NR2B significantly more sensitive to ethanol than NR2C and NR2D. (3) Manipulation of the NMDA receptor with a number of agents that normally modulate it did not alter the degree of inhibition produced by ethanol. The presence of Mg²⁺ (3 and 12.5 µ M ), Zn²⁺ (1 and 10 µ M ), or the glycine antagonist 7-chlorokynurenic acid (1.25 or 5 µ M ), did not alter the ethanol sensitivity of heteromeric (NR1b/NR2A, NR1b/NR2B, NR1b/NR2C) NMDA receptors. Redox modulation of the NMDA receptor by dithiothreitol (2 m M ) or 5,5'-dithiobis(2-nitrobenzoic acid) (1 m M ) also did not alter the degree to which ethanol inhibits NMDA receptors. Taken together, these results indicate that the ethanol sensitivity of native NMDA receptors, which likely exist in heteromeric form, results from actions at a site different from those of known modulators of the receptor.     

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.     

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.     

Mechanisms of activation,inhibition and specificity: crystal structures of the NMDA receptor NR1 ligand-binding core

Excitatory neurotransmission mediated by the N-methyl-D-aspartate subtype of ionotropic glutamate receptors is fundamental to the development and function of the mammalian central nervous system. NMDA receptors require both glycine and glutamate for activation with NR1 and NR2 forming glycine and glutamate sites, respectively. Mechanisms to describe agonist and antagonist binding, and activation and desensitization of NMDA receptors have been hampered by the lack of high-resolution structures. Here, we describe the cocrystal structures of the NR1 S1S2 ligand-binding core with the agonists glycine and D-serine (DS), the partial agonist D-cycloserine (DCS) and the antagonist 5,7-dichlorokynurenic acid (DCKA). The cleft of the S1S2 'clamshell' is open in the presence of the antagonist DCKA and closed in the glycine, DS and DCS complexes. In addition, the NR1 S1S2 structure reveals the fold and interactions of loop 1, a cysteine-rich region implicated in intersubunit allostery.     

Activation of N-Methyl-d-aspartate (NMDA) Receptors in the Dorsal Vagal Complex Lowers Glucose Production

Diabetes is characterized by hyperglycemia due partly to increased hepatic glucose production. The hypothalamus regulates hepatic glucose production in rodents. However, it is currently unknown whether other regions of the brain are sufficient in glucose production regulation. The N-methyl-d-aspartate (NMDA) receptor is composed of NR1 and NR2 subunits, which are activated by co-agonist glycine and glutamate or aspartate, respectively. Here we report that direct administration of either co-agonist glycine or NMDA into the dorsal vagal complex (DVC), targeting the nucleus of the solitary tract, lowered glucose production in vivo. Direct infusion of the NMDA receptor blocker MK-801 into the DVC negated the metabolic effect of glycine. To evaluate whether NR1 subunit of the NMDA receptor mediates the effect of glycine, NR1 in the DVC was inhibited by DVC NR1 antagonist 7-chlorokynurenic acid or DVC shRNA-NR1. Pharmacological and molecular inhibition of DVC NR1 negated the metabolic effect of glycine. To evaluate whether the NMDA receptors mediate the effects of NR2 agonist NMDA, DVC NMDA receptors were inhibited by antagonist d-2-amino-5-phosphonovaleric acid (d-APV). DVC d-APV fully negated the ability of DVC NMDA to lower glucose production. Finally, hepatic vagotomy negated the DVC glycine ability to lower glucose production. These findings demonstrate that activation of NR1 and NR2 subunits of the NMDA receptors in the DVC is sufficient to trigger a brain-liver axis to lower glucose production, and suggest that DVC NMDA receptors serve as a therapeutic target for diabetes and obesity.     

Intersubunit cooperativity in the NMDA receptor.

Opening of the NMDA receptor channel requires simultaneous binding of glutamate and glycine. Although the binding sites for each agonist are in different subunits, the presence of one agonist influences the binding of the other. We have localized regions in the S1 binding domain of both subunits required for the transmission of allosteric signals from the glutamate binding NR2A subunit to the glycine binding NR1 subunit. Three-dimensional modeling indicates that these segments are not directly involved in ligand binding, but likely form solvent-accessible loops protruding out of the binding pocket, making them suitable to relay interactions between adjacent subunits. Thus, these segments mediate negative allosteric coupling between the two subunit types that form the NMDA receptor.     

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.     

Fyn tyrosine kinase reduces the ethanol inhibition of recombinant NR1/NR2A but not NR1/NR2B NMDA receptors expressed in HEK 293 cells

NMDA receptors are potentiated by phosphorylation in a subunit- and kinase-specific manner. Both native and recombinant NMDA receptors are inhibited by behaviorally relevant concentrations of ethanol. Whether the phosphorylation state of individual subunits modulates the ethanol sensitivity of these receptors is not known. In this study, the effects of Fyn tyrosine kinase on the ethanol sensitivity of specific recombinant NMDA receptors expressed in HEK 293 cells were investigated. Whole-cell mode patch clamp and ratiometric calcium imaging demonstrated that the degree of ethanol inhibition of NR1/NR2B receptors was unaffected by Fyn tyrosine kinase. In contrast, the inhibition of NR1/NR2A receptors by ethanol (100 mM) was significantly reduced under conditions of enhanced Fyn-mediated tyrosine phosphorylation of the NR2A subunit. This effect was not observed at lower concentrations of ethanol (< or = 50 mM). These results suggest that tyrosine phosphorylation of specific NMDA receptors by Fyn tyrosine kinase may regulate the sensitivity of these receptors to the sedative/hypnotic concentrations of ethanol.     

Interactions among Positions in the Third and Fourth Membrane-associated Domains at the Intersubunit Interface of the N-Methyl-D-aspartate Receptor Forming Sites of Alcohol Action

The N-methyl-d-aspartate (NMDA) glutamate receptor is a major target of ethanol in the brain. Previous studies have identified positions in the third and fourth membrane-associated (M) domains of the NMDA receptor GluN1 and GluN2A subunits that influence alcohol sensitivity. The predicted structure of the NMDA receptor, based on that of the related GluA2 subunit, indicates a close apposition of the alcohol-sensitive positions in M3 and M4 between the two subunit types. We tested the hypothesis that these positions interact to regulate receptor kinetics and ethanol sensitivity by using dual substitution mutants. In single-substitution mutants, we found that a position in both subunits adjacent to one previously identified, GluN1(Gly-638) and GluN2A(Phe-636), can strongly regulate ethanol sensitivity. Significant interactions affecting ethanol inhibition and receptor deactivation were observed at four pairs of positions in GluN1/GluN2A: Gly-638/Met-823, Phe-639/Leu-824, Met-818/Phe-636, and Leu-819/Phe-637; the latter pair also interacted with respect to desensitization. Two interactions involved a position in M4 of both subunits, GluN1(Met-818) and GluN2A(Leu-824), that does not by itself alter ethanol sensitivity, whereas a previously identified ethanol-sensitive position, GluN2A(Ala-825), did not unequivocally interact with any other position tested. These results also indicate a shift by one position of the predicted alignment of the GluN1 M4 domain. These findings have allowed for the refinement of the NMDA receptor M domain structure, demonstrate that this region can influence apparent agonist affinity, and support the existence of four sites of alcohol action on the NMDA receptor, each consisting of five amino acids at the M3-M4 domain intersubunit interfaces.     

Single-channel conductances of NMDA receptors expressed from cloned cDNAs: comparison with native receptors.

To cast light on the subunit composition of native NMDA-type glutamate receptors, four cloned subunits of the NMDA receptor have been expressed, in pairs, in Xenopus oocytes, and their single-channel properties have been measured. The conductances of the channels, and their characteristic patterns of sublevel transitions, turn out to be useful diagnostic criteria for subunit composition. The NR1-NR2A and NR1-NR2B combinations (which have identical TM2 sequences) are very similar to each other. Both have 50 pS openings and brief 40 pS sublevels (in 1 mM external Ca2+), with similar mean lifetimes and frequencies. They also show close quantitative resemblance to the channels of hippocampal CA1 and dentate gyrus cells and of cerebellar granule cells, except that the NR1-NR2A combination has a lower glycine sensitivity than the native channels. In contrast, the NR1-NR2C combination produces a channel with 36 pS and 19 pS conductances of similar (brief) duration; these closely resemble the 38-18 pS channels that have previously been observed in large cerebellar neurons in culture (together with 50 pS channels).     

An Endoplasmic Reticulum Retention Signal Located in the Extracellular Amino-terminal Domain of the NR2A Subunit of N-Methyl-d-aspartate Receptors

N-Methyl-d-aspartate (NMDA) receptors play critical roles in complex brain functions as well as pathogenesis of neurodegenerative diseases. There are many NMDA isoforms and subunit types that, together with subtype-specific assembly, give rise to significant functional heterogeneity of NMDA receptors. Conventional NMDA receptors are obligatory heterotetramers composed of two glycine-binding NR1 subunits and two glutamate-binding NR2 subunits. When individually expressed in heterogeneous cells, most of the NR1 splice variants and the NR2 subunits remain in the endoplasmic reticulum (ER) and do not form homomeric channels. The mechanisms underlying NMDA receptor trafficking and functional expression remain uncertain. Using truncated and chimeric NMDA receptor subunits expressed in heterogeneous cells and hippocampal neurons, together with immunostaining, biochemical, and functional analyses, we found that the NR2A amino-terminal domain (ATD) contains an ER retention signal, which can be specifically masked by the NR1a ATD. Interestingly, no such signal was found in the ATD of the NR2B subunit. We further identified the A2 segment of the NR2A ATD to be the primary determinant of ER retention. These findings indicate that NR2A-containing NMDA receptors may undergo a different ER quality control process from NR2B-containing NMDA receptors.Ionotropic glutamate receptors (iGluRs)² mediate most of the excitatory neurotransmission in the central nervous system. They play key roles in complex brain functions as well as in the pathogenesis of neurodegenerative diseases. Based on pharmacological properties and sequence similarities, iGluRs can be grouped into three major subtypes: GluR1 to -4 subunits form α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors, GluR5 to -7 and KA1 and -2 subunits make up kainate receptors, and NR1 together with NR2A to -D subunits comprise the NMDA receptors (1). All iGluR subunits share a unique membrane topology with a large extracellular NH₂-terminal domain, three transmembrane segments (TM1 (transmembrane domain 1), TM3, and TM4), a P-loop region, and a cytoplasmic COOH terminus (2, 3). Based on the sequence homology to bacterial periplasmic binding proteins, the NH₂-terminal domain of iGluRs can be divided into two domains in tandem: the amino-terminal domain (ATD), which includes the first 400 or so amino acids (4), and the following S1 domain preceding TM1, which forms the ligand-binding domain together with the extracellular loop between TM3 and TM4 (S2 domain) (5, 6).Among iGluRs, NMDA receptors are special in that conventional NMDA receptors are obligatory tetrameric membrane proteins composed of two glycine-binding NR1 and two glutamate-binding NR2 subunits. The NR1 subunit is essential for the formation of functional NMDA receptor channel, whereas the NR2 subunit modifies channel properties, such as current kinetics and channel conductance (1). The major NR1 splice variant and the NR2 subunits are retained in the ER when expressed alone in heterogeneous cells. Only when expressed together do they form functional receptors on the cell surface (7–9). In the last decade, enormous progress has been made in understanding the phenomenology and mechanisms of functional plasticity of NMDA receptors. However, much less is known about the mechanisms underlying the ER retention of NMDA receptor subunits. Previous studies focused on the COOH terminus have shown that the NR1a subunit contains an ER retention signal, RRR, in the C1 cassette, whereas a motif, HLFY, found in the NR2B subunit immediately following the TM4 (10) or, at least, the presence of any two amino acid residues after NR2 TM4 (11) is required for the export of NR1-NR2 complexes from the ER. Recently, novel ER retention signals were identified in the TM3 of both NR1 and NR2B subunits. In addition, TM3 of both NR1 and NR2B and TM4 of NR1 are necessary for masking ER retention signals found in TM3 (12).In the present study, we focused on the functional role of the ATD in the surface expression of NMDA receptors. Interestingly, we found an ER retention signal located in the ATD of the NR2A subunit but not in the corresponding domain of the NR2B. It is suggested that NR2A-containing NMDA receptors may undergo an ER quality control process different from that of NR2B-containing NMDA receptors.     

Toxicity of beta-amyloid in HEK293 cells expressing NR1/NR2A or NR1/NR2B N-methyl-D-aspartate receptor subunits

Neurotoxicity induced by beta-amyloid peptide (Abeta) involves glutamate toxicity, resulting from overactivation of N-methyl-D-aspartate (NMDA) receptors and elevation of intracellular calcium. However, the heterogeneity of the NMDA receptors, frequently composed of NR1 and NR2A-D subunits, has been less studied. Thus, we determined the contribution of NMDA receptor subtypes on Abeta(1-40) toxicity in HEK293 cells transiently expressing NR1/NR2A or NR1/NR2B subunits. Analysis of lactate dehydrogenase (LDH) release and trypan blue exclusion revealed an increase in Abeta(1-40) toxicity upon NR1/NR2A expression, compared to NR1/NR2B, indicating loss of plasma membrane integrity. Furthermore, Abeta(1-40) decreased intracellular ATP in cells expressing NR1/NR2A. MK-801 ((+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate), a noncompetitive NMDA receptor antagonist, partially prevented the decrease in cell viability and the energy impairment. These differences were not accounted for by the activation of caspases 2, 3, 8 and 9 or calpains or by DNA fragmentation, excluding the hypothesis of apoptosis. Functional NR1/NR2A and NR1/NR2B receptor subtypes were further evidenced by single-cell calcium imaging. Stimulation of NR1/NR2A receptors with NMDA/glycine revealed an increase in intracellular calcium in cells pre-exposed to Abeta(1-40). Opposite effects were observed upon activation of NR1/NR2B receptors. These results suggest that NR1/NR2A-composed NMDA receptors mediate necrotic cell death in HEK293 cells exposed to Abeta(1-40) through changes in calcium homeostasis.     

A three amino acid tail following the TM4 region of the N-methyl-D-aspartate receptor (NR) 2 subunits is sufficient to overcome endoplasmic reticulum retention of NR1-1a subunit

The cytoplasmic C-terminal domains of NR2 subunits have been proposed to modulate the assembly and trafficking of NMDA receptors. However, questions remain concerning which domains in the C terminus of NR2 subunits control the assembly of receptor complexes and how the assembled complexes are selectively trafficked through the various cellular compartments such as endoplasmic reticulum (ER) to the cell surface. In the present study, we found that the three amino acid tail after the TM4 region of NR2 subunits is necessary for surface expression of functional NMDA receptors, while truncations with only two amino acids following the TM4 region (NR2Delta2) completely eliminated surface expression of the NMDA receptor on co-expression with NR1-1a in HEK293 cells. FRET (fluorescence resonance energy transfer) analysis showed that these NR2Delta2 truncations are able to form homomers and heteromers on co-expression with NR1-1a. Furthermore, when NR2Delta2 subunits were cotransfected with either the NR1-4a or NR1-1a(AAA) mutant, lacking the ER retention motif (RRR), functional NMDA receptors were detected in the transfected HEK293 cells. Unexpectedly, we found that the replacement of five residues after TM4 with alanines gave results indistinguishable from those of NR2BDelta5 (EHLFY), demonstrating the short tail following the TM4 of NR2 subunits is not sequence-specific-dependent. Taken together, our results show that the C terminus of the NR2 subunits is not necessary for the assembly of NMDA receptor complexes, whereas a three amino acid long cytoplasmic tail following the TM4 of NR2 subunits is sufficient to overcome the ER retention existing in the C terminus of NR1, allowing the assembled NMDA receptors to reach the cell surface.     

N-methyl-D-aspartate receptor-dependent regulation of the glutamate transporter excitatory amino acid carrier 1

The neuronal transporter excitatory amino acid carrier 1 (EAAC1) is enriched in perisynaptic regions, where it may regulate synaptic spillover of glutamate. In this study we examined potential interactions between EAAC1 and ionotropic glutamate receptors. N-Methyl-D-aspartate (NMDA) receptor subunits NR1, NR2A, and NR2B, but not the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor subunit GluR2, were co-immunoprecipitated with EAAC1 from neuron-enriched hippocampal cultures. A similar interaction was observed in C6 glioma and human embryonic kidney cells after co-transfection with Myc epitope-tagged EAAC1 and NMDA receptor subunits. Co-transfection of C6 glioma with the combination of NR1 and NR2 subunits dramatically increased (approximately 3-fold) the amount of Myc-EAAC1 that can be labeled with a membrane-impermeable biotinylating reagent. In hippocampal cultures, brief (5 min), robust (100 microM NMDA, 10 microM glycine) activation of the NMDA receptor decreased biotinylated EAAC1 to approximately 50% of control levels. This effect was inhibited by an NMDA receptor antagonist, intracellular or extracellular calcium chelators, or hypertonic sucrose. Glutamate, alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid with cyclothiazide, and thapsigargin mimicked the effects of NMDA. These studies suggest that NMDA receptors interact with EAAC1, facilitate cell surface expression of EAAC1 under basal conditions, and control internalization of EAAC1 upon activation. This NMDA receptor-dependent regulation of EAAC1 provides a novel mechanism that may shape excitatory signaling during synaptic plasticity and/or excitotoxicity.     

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.     

Chronic ethanol exposure delays the 'developmental switch' of the NMDA receptor 2A and 2B subunits in cultured cerebellar granule neurons

Chronic ethanol treatment of cultured neurons from various brain areas has been found to increase NMDA receptor function and to alter the levels of some NMDA receptor subunit proteins. Because the cultured neurons are exposed to ethanol during a period when the NMDA receptor is undergoing developmental changes in subunit expression, we wished to determine whether ethanol treatment alters this developmental pattern. We found that 3 days of treatment of cerebellar granule neurons with ethanol, which was previously reported to increase NMDA receptor function, resulted in a delay in the 'developmental switch' of the NR2A and NR2B subunits, i.e. the developmental decrease in NR2B and increase in NR2A protein expression. As a result, the level of NR2B was higher, and that of NR2A was lower, in the ethanol-treated cells than in control cells. Cross-linking experiments showed that the changes in total receptor subunit proteins levels were reflected in cell-surface expressed proteins, indicating changes in the amount of functional receptors. These results were confirmed by a higher potency of glycine at the NMDA receptor in the ethanol-treated cells, as determined by NMDA/glycine-induced increases in intracellular Ca(2+). The results suggest that the mechanism by which ethanol alters NMDA receptor expression in cultured neurons, where receptors are undergoing development, differs from the mechanism of ethanol's effect on NMDA receptors in adult brain. Changes in the proportion of NR2A and NR2B subunits may contribute to effects of ethanol on neuronal development.     

Model structures of the N-methyl-D-aspartate receptor subunit NR1 explain the molecular recognition of agonist and antagonist ligands

Molecular models of the ligand-binding domain of N-methyl-d-aspartate subunit R1 (NR1) were made using the published crystal structures of rat glutamate receptor B (GluRB), the bacterial glutamate receptor (GluR0), and the glutamine-binding protein (QBP) of Escherichia coli. Separate models of NR1 were built to represent the ligand-binding conformation for agonist (glycine, d- and l-isomers of serine and alanine, and the partial agonist ligand d-cycloserine) and antagonist (5,7-dichloro-4-oxo-1,4-dihydroquinoline-2-carboxylic acid (DCKA) and E-3-(2-phenyl-2-carboxyethenyl)-4,6-dichloro-1-H-indole-2-carboxylic acid (MDL 105,519)) ligands. Side-chain conformations of residues within the NR1 ligand-binding site were selected that optimized the hydrophobic packing and hydrogen bonding among residues, while taking into account published data comparing receptor mutants with wild-type NR1. Ligands docked to the model structures provide a rational explanation for the observed differences in binding affinity and receptor activation among agonist and antagonist ligands. NR1 prefers smaller ligands (glycine, serine, and alanine) in comparison with GluRB and GluR0 that bind l-glutamate: the bulky side chain of W731 in NR1 dramatically reduces the size of the ligand-binding site, functioning to selectively restrict recognition to glycine and the d-isomers of serine and alanine. Nevertheless, many of the interactions seen for ligands bound to GluRB, GluR0, and periplasmic-binding proteins are present for the ligands docked to the model structures of NR1.