Sites in the fourth membrane-associated domain regulate alcohol sensitivity of the NMDA receptor

N-methyl-D-aspartate (NMDA) receptors are important target sites of alcohol action in the central nervous system. Alcohol inhibits NMDA receptor current by an action on ion channel gating, apparently through a direct action on a region of the NMDA receptor accessible from the extracellular environment. Our previous studies have revealed an important role for a methionine residue (Met823) in membrane-associated domain 4 (M4) of the NR2A subunit in channel gating as well as alcohol sensitivity of the NMDA receptor. The role of sites in M4 of the NMDA receptor NR2A subunit adjacent to Met823 was investigated using tryptophan-scanning mutagenesis and electrophysiological recording. Receptors containing NR1 and NR2A(V820W) or NR2A(M817W) mutant subunits expressed in HEK 293 cells were not functional. The mutation Ala826Trp modified apparent desensitization, and the mutations Ala825Trp and Ala826Trp changed the mean open time of the channel as determined by fluctuation analysis. In addition, the mutations Tyr822Trp and Ala825Trp significantly altered the concentration-response curves for ethanol inhibition. The changes in mean open time did not appear to be able to account for the observed differences in ethanol sensitivity. These results indicate that this region in M4 of the NR2A subunit may be involved in the action of alcohol.     

Molecular determinants of proton-sensitive N-methyl-D-aspartate receptor gating

Extracellular protons inhibit N-methyl-D-aspartate (NMDA) receptors with an IC50 value in the physiological pH range. To identify the molecular determinants of proton sensitivity, we used scanning mutagenesis of the NR1 subunit to search for residues that control proton inhibition of NMDA receptors. Homology modeling of the extracellular domains suggested that residues at which mutations perturbed pH sensitivity were localized in discrete regions. The majority of mutations that strongly affected proton sensitivity were clustered in the extracellular end of the second transmembrane domain (M3) and adjacent linker leading to the S2 portion of the glycine-binding domain of NR1. Mutations in NR2A confirmed that the analogous region controls the pH sensitivity of this subunit and also identified the linker region between the third transmembrane domain (M4) and the S2 portion of the NR2 glutamate binding domain as an additional determinant of proton sensitivity. One mutant receptor, NR1(A649C)/NR2A(A651T), showed a 145-fold reduction in the IC50 for protons (IC50, 17.3 microM corresponding to pH 4.9). The M3-S2 linker region has been suggested to control NMDA receptor gating, leading to the hypothesis that the proton sensor and receptor gate may be structurally and functionally integrated.     

Alcohols inhibit N-methyl-D-aspartate receptors via a site exposed to the extracellular environment

N-Methyl-D-aspartate (NMDA) receptors are important CNS target sites of alcohols, but the site and mechanism of action of alcohols on NMDA receptors remains unclear. In CHO-K1 cells transfected with NR1/NR2B NMDA receptor subunits, ethanol inhibited NMDA-activated current with an IC(50) of 138 mM. Truncation of the intracellular C-terminal domain of the NR1 subunit (NR1T) did not alter ethanol sensitivity when combined with the NR2B subunit, but a similar truncation of the NR2B subunit (NR2BT) slightly enhanced ethanol sensitivity of receptors formed from coexpression with either NR1 or NR1T subunits. 1-Pentanol applied externally inhibited NMDA receptors with an IC(50) of 9.9 mM, but intracellular application of 1-pentanol (25 mM) did not alter NMDA receptor inhibition by externally applied ethanol or 1-pentanol. In addition, the amplitude of NMDA-activated current did not decrease during the time required for 1-pentanol (25 mM) to diffuse throughout the cytoplasm. Ethanol did not inhibit NMDA receptors when bath-applied in cell-attached patches or when applied to the cytoplasmic face of inside-out membrane patches. These results appear to be best explained by an action of alcohols on the NMDA receptor-channel protein, at a site located in a domain exposed to, or only accessible from, the extracellular environment.     

Probing N-methyl-D-aspartate receptor desensitization with the substituted-cysteine accessibility method

Several forms of macroscopic N-methyl-D-aspartate (NMDA) receptor desensitization affect the amplitude and duration of postsynaptic responses. In addition to its functional significance, desensitization provides one means to examine the conformational coupling of ligand binding to channel gating. Segments flanking the ligand binding domain in the extracellular N terminus of the NMDA receptor NR2 subunit influence the glycine-independent form of desensitization. The NR2A pre-M1 region, the linker between the glutamate binding domain and the channel pore, plays a critical role in desensitization. Thus, we used the substituted-cysteine accessibility method to scan the accessibility of residues in the pre-M1 region and the first transmembrane domain (M1) of NR2A. Cysteine mutants were expressed with NR1 in human embryonic kidney 293 cells and were assayed by whole-cell recording. With activation of the receptor by glutamate and glycine, only a single mutant, V557C, which is located at the beginning of M1, led to irreversible inhibition by the methanethiosulfonate derivative methanethiosulfonate ethyltrimethylammonium (MTSET). The NR2 ligand glutamate was insufficient on its own to induce modification of V557C by MTSET, suggesting that the change in accessibility required channel gating. The rate of MTSET modification of the homologous residue on NR1 (NR1-1a(L562C)/NR2A) was much slower than V557C. We also substituted cysteine in the V557 site of mutant subunits that exhibit either enhanced or reduced desensitization. Modification by MTSET correlated with the degree of desensitization for these subunits, suggesting that V557C is a sensitive detector of desensitization gating.     

Site within N-Methyl-D-aspartate receptor pore modulates channel gating

N-methyl-d-aspartate-type glutamate receptors (NMDARs) are ligand-gated ion channels activated by coagonists glutamate and glycine. NMDARs play a critical role in synaptic plasticity and excitotoxicity, largely because of their high calcium permeability and slow deactivation and desensitization kinetics. NR1 is an obligate subunit in all NMDAR complexes, where it combines with NR2A, 2B, 2C, and/or 2D. NR1 binds glycine, and residue Asn598 in the re-entrant membrane loop M2 largely determines NMDAR calcium permeability. In contrast, NR2 subunits bind glutamate and contain regions that regulate receptor desensitization and deactivation. Here, we report that mutations of NR1(Asn598) in combination with wild-type NR2A, expressed in human embryonic kidney 293 cells, exhibit altered glycine-independent desensitization. In the absence of extracellular calcium, substitution of Arg for Asn598 (NR1R) slowed desensitization by 2- to 3-fold compared with wild-type NR1/NR2A, and glutamate-evoked peak current EC50 and deactivation rate were also affected. Replacement of Asn by Gln (NR1Q) produced two distinct rates of calcium- and glycine-independent desensitization. Moreover, in the presence of extracellular calcium, the voltage-dependent pore block by calcium for the NR1Q mutant mimicked the effects of the positively charged Arg at this site in NR1R on slowing desensitization and deactivation. A kinetic model of the NMDA receptor-channel suggests that these results can be explained by altered gating and not ligand binding. Our data increase understanding of the role that amino acids within the NMDAR pore play in channel gating.     

Differential actions of ethanol and trichloroethanol at sites in the M3 and M4 domains of the NMDA receptor GluN2A (NR2A) subunit

Background and purpose:

Alcohol produces its behavioural effects in part due to inhibition of N-methyl-d-aspartate (NMDA) receptors in the CNS. Previous studies have identified amino acid residues in membrane-associated domains 3 (M3) and 4 (M4) of the NMDA receptor that influence ethanol sensitivity. In addition, in other alcohol-sensitive ion channels, sedative-hypnotic agents have in some cases been shown to act at sites distinct from the sites of ethanol action. In this study, we compared the influence of mutations at these sites on sensitivity to ethanol and trichloroethanol, a sedative-hypnotic agent that is a structural analogue of ethanol.

Experimental approach:

We constructed panels of mutants at ethanol-sensitive positions in the GluN2A (NR2A) NMDA receptor subunit and transiently expressed these mutants in human embryonic kidney 293 cells. We used whole-cell patch-clamp recording to assess the actions of ethanol and trichloroethanol in these mutant NMDA receptors.

Key results:

Ethanol sensitivity of mutants at GluN2A(Ala825) was not correlated with any physicochemical measures tested. Trichloroethanol sensitivity was altered in two of three ethanol-insensitive mutant GluN2A subunits: GluN2A(Phe637Trp) in M3 and GluN2A(Ala825Trp) in M4, but not GluN2A(Met823Trp). Trichloroethanol sensitivity decreased with increasing molecular volume at Phe637 or increasing hydrophobicity at Ala825 and was correlated with ethanol sensitivity at both sites.

Conclusions and implications:

Evidence obtained to date is consistent with a role of GluN2A(Ala825) as a modulatory site for ethanol and trichloroethanol sensitivity, but not as a binding site. Trichloroethanol appears to inhibit the NMDA receptor in a manner similar, but not identical to, that of ethanol.     

Equilibrium constants for (R)-[(S)-1-(4-bromo-phenyl)-ethylamino]-(2,3-dioxo-1,2,3,4-tetrahydroquinoxalin-5-yl)-methyl]-phosphonic acid (NVP-AAM077) acting at recombinant NR1/NR2A and NR1/NR2B N-methyl-D-aspartate receptors: Implications for studies of synaptic transmission

We have quantified the effects of the N-methyl-d-aspartate (NMDA) receptor antagonist (R)-[(S)-1-(4-bromo-phenyl)-ethylamino]-(2,3-dioxo-1,2,3,4-tetrahydroquinoxalin-5-yl)-methyl]-phosphonic acid (NVP-AAM077) at rat recombinant N-methyl-D-aspartate receptor (NR)1/NR2A and NR1/NR2B NMDA receptors expressed in Xenopus laevis oocytes. We observed no difference in the steady-state levels of inhibition produced by NVP-AAM077 when it was either preapplied or coapplied with glutamate. The IC50 values for NVP-AAM077 acting at NR1/NR2A NMDA receptors were, as expected, dependent on the glutamate concentration used to evoke responses, being 31 +/- 2 nM (with glutamate at its EC50 concentration) and 214 +/- 10 nM (at 10 times the EC50 concentration). Schild analysis confirmed that the antagonism produced by NVP-AAM077 at NR1/NR2A NMDA receptors was competitive and gave an estimate of its equilibrium constant (K(B)) of 15 +/- 2 nM. Furthermore, Schild analysis of an NMDA receptor carrying a threonine-to-alanine point mutation in the NR2A ligand binding site indicated that NVP-AAM077 still acted in a competitive manner but with its K(B) increased by around 15-fold. At NR1/NR2B NMDA receptors, NVP-AAM077 displayed reduced potency. An IC50 value of 215 +/- 13 nM was obtained in the presence of the EC50 concentration of glutamate (1.5 microM), whereas a value of 2.2 +/- 0.14 microM was obtained with higher (15 microM) glutamate concentrations. Schild analysis gave a K(B) for NVP-AAM077 at NR2B-containing receptors of 78 +/- 3 nM. Finally, using a kinetic scheme to model "synaptic-like" activation of NMDA receptors, we show that the difference in the equilibrium constants for NVP-AAM077 is not sufficient to discriminate between NR2A-containing or NR2B-containing NMDA receptors.     

Adenosine triphosphate acts as both a competitive antagonist and a positive allosteric modulator at recombinant N-methyl-D-aspartate receptors

ATP and glutamate are fast excitatory neurotransmitters in the central nervous system acting primarily on ionotropic P2X and glutamate [N-methyl-D-aspartate (NMDA) and non-NMDA] receptors, respectively. Both neurotransmitters regulate synaptic plasticity and long-term potentiation in hippocampal neurons. NMDA receptors are responsible primarily for the modulatory action of glutamate, but the mechanism underlying the modulatory effect of ATP remains uncertain. In the present study, the effect of ATP on recombinant NR1a + 2A, NR1a + 2B, and NR1a + 2C NMDA receptors expressed in Xenopus laevis oocytes was investigated. ATP inhibited NR1a + 2A and NR1a + 2B receptor currents evoked by low concentrations of glutamate but potentiated currents evoked by saturating glutamate concentrations. In contrast, ATP potentiated NR1a + 2C receptor currents evoked by nonsaturating glutamate concentrations. ATP shifted the glutamate concentration-response curve to the right, indicating a competitive interaction at the agonist binding site. ATP inhibition and potentiation of glutamate-evoked currents was voltage-independent, indicating that ATP acts outside the membrane electric field. Other nucleotides, including ADP, GTP, CTP, and UTP, inhibited glutamate-evoked currents with different potencies, revealing that the inhibition is dependent on both the phosphate chain and nucleotide ring structure. At high concentrations, glutamate outcompetes ATP at the agonist binding site, revealing a potentiation of the current. This effect must be caused by ATP binding at a separate site, where it acts as a positive allosteric modulator of channel gating. A simple model of the NMDA receptor, with ATP acting both as a competitive antagonist at the glutamate binding site and as a positive allosteric modulator at a separate site, reproduced the main features of the data.     

Mutations within the selectivity filter of the NMDA receptor-channel influence voltage dependent block by 5-hydroxytryptamine

BACKGROUND AND PURPOSE: Voltage-dependent block by Mg2+ is a cardinal feature of NMDA receptors which acts as a coincidence detector to prevent the receptor from over-activation. Inhibition of NMDA receptor currents by 5-hydroxytryptamine (5-HT) indicated that 5-HT, similar to Mg2+, binds within the membrane electric field. In the present study, we assessed whether point mutations of critical asparagine residues located within the selectivity filter of NR1 and NR2A subunits of NMDA receptor-channel affect voltage-dependent block by 5-HT. EXPERIMENTAL APPROACH: The mode of action of 5-HT and Mg2+ on wild-type and mutated NMDA receptor-channels expressed in Xenopus oocytes was investigated using the two-electrode voltage clamp recording technique. KEY RESULTS: The mutation within the NR1 subunit NR1(N0S or N0Q) strongly reduced the voltage dependent block by 5-HT and increased the IC50. The corresponding mutations within the NR2 subunits NR2A(N0Q or N+1Q) reduced the block by 5-HT to a lesser extent. This is in contrast to the block produced by external Mg2+ where a substitution at the NR2A(N0) and NR2A(N+1) sites but not at the NR1(N0) site significantly reduced Mg2+ block.CONCLUSION AND IMPLICATIONS: The block of NMDA receptor-channels by 5-HT depends on the NR1-subunit asparagine residue and to a lesser extent on the NR2A-subunit asparagine residues. These data suggest that the interaction of 5-HT with functionally important residues in a narrow constriction of the pore of the NMDA receptor-channel provides a significant barrier to ionic fluxes through the open channel due to energetic factors governed by chemical properties of the binding site and the electric field.     

The NR2B subtype of NMDA receptor: a potential target for the treatment of alcohol dependence

Ethanol is a small molecule acting on several neurotransmitter systems in the brain. Accumulating evidences suggest that the primary excitatory--i.e. the glutamatergic--neurotransmitter system is a particularly important site of ethanol's action. Several studies showed that ethanol is a potent and selective inhibitor of the N-methyl-D-aspartate (NMDA) receptors and prolonged ethanol exposition leads to a compensatory "up-regulation" of these receptors resulting in enhanced NMDA receptor-mediated functions after removal of ethanol. These alterations are supposed to contribute to the development of ethanol tolerance, dependence as well as the acute and delayed signs of ethanol withdrawal. In recent papers, alterations in subunit composition of NMDA receptors were reported after long term ethanol exposure. mRNA and/or protein levels of NR2A and NR2B types of subunits were found elevated both by in vivo and in vitro experiments. Our results showed that especially the NR2B subunit expression is increased in cultured hippocampal and cortical neurones after 3 days of intermittent ethanol treatment. According to the high calcium permeability, the increased agonist sensitivity and the relatively slow closing kinetics of NMDA ion channels composed of NR2B subunits, the above mentioned changes may underlie the enhanced NMDA receptor activation observed after long term ethanol exposure. Accordingly, we have tested NR2B subunit selective NMDA receptor antagonists in primary cultures of rat cortical neurones pre-treated with ethanol intermittently for 3 days and found that these compounds potently inhibited the neurotoxic effect of ethanol withdrawal. Hypothesising the involvement of enhanced NR2B subunit expression in development of alcohol dependence and withdrawal symptoms and considering the tolerable side effect profile of the NR2B subunit selective NMDA receptor antagonists, the NR2B type of NMDA receptor subunit may serve as a possible drug target in pharmacological interventions for alcoholism. The aim of this review is to give an update on the role of altered structure and function of NMDA receptors after ethanol exposure and to summarise the recent data about the activity of NR2B subunit selective NMDA receptor antagonists in model systems related to alcoholism.     

Role of altered structure and function of NMDA receptors in development of alcohol dependence

Long-term alcohol exposure gives rise to development of physical dependence on alcohol in consequence of changes in certain neurotransmitter functions. Accumulating evidence suggests that the glutamatergic neurotransmitter system, especially the N-methyl-D-aspartate (NMDA) type of glutamate receptors is a particularly important site of ethanol's action, since ethanol is a potent inhibitor of the NMDA receptors (NMDARs) and prolonged ethanol exposition leads to a compensatory "upregulation" of NMDAR mediated functions supposedly contributing to the occurrence of ethanol tolerance, dependence as well as the acute and delayed signs of ethanol withdrawal.Recently, expression of different types of NMDAR subunits was found altered after long-term ethanol exposure. Especially, the expression of the NR2B and certain splice variant forms of the NR1 subunits were increased in primary neuronal cultures treated intermittently with ethanol. Since NMDA ion channels with such an altered subunit composition have increased permeability for calcium ions, increased agonist sensitivity, and relatively slow closing kinetics, the abovementioned alterations may underlie the enhanced NMDAR activation observed after long-term ethanol exposure. In accordance with these changes, the inhibitory potential of NR2B subunit-selective NMDAR antagonists is also increased, demonstrating excellent potency against alcohol withdrawal-induced in vitro cytotoxicity. Although in vivo data are few with these compounds, according to the effectiveness of the classic NMDAR antagonists in attenuation, not only the physical symptoms, but also some affective and motivational components of alcohol withdrawal, novel NR2B subunit selective NMDAR antagonists may offer a preferable alternative in the pharmacotherapy of alcohol dependence.     

Antagonists selective for NMDA receptors containing the NR2B subunit

In the late 1980s, a new class of N-methyl-D-aspartate (NMDA) receptor antagonists, exemplified by the phenylethanolamine ifenprodil (1), was identified. Initially, the mechanism of action of ifenprodil was a mystery as it was not a competitive antagonist at the glutamate or glycine (co-agonist) binding sites, nor was it a blocker of the calcium ion channel associated with the NMDA receptor. Early studies with a novel polyamine binding site associated with the NMDA receptor and functional studies in various brain regions suggested a unique and selective activity profile for 1. However, it was not until the NMDA receptor subunits were identified and expressed that ifenprodil was shown to be a selective antagonist for a subset of NMDA receptors containing the NR2B subunit. The wide range of potential therapeutic targets for NMDA antagonists coupled with the hope that NR2B selective agents might possess an improved clinical safety profile compared to non-selective compounds has supported an aggressive effort to develop the structure-activity relationships (SAR) of NR2B selective antagonists. This SAR and the basic physiology of the NMDA receptor form the basis of this review.     

Influence of external magnesium ions on the NMDA receptor channel block by different types of organic cations

The NMDA type of ionotropic glutamate receptors plays a unique role in synaptic functions because of high permeability for calcium and because of a voltage-dependent block by endogenous Mg(2+). Activity and voltage dependence of the NMDA receptor channel block by organic cations are strongly affected by competition with magnesium ions for the binding site in the channel pore. It complicates prediction of action of NMDA receptor channel blockers in vivo. In the present work we studied the NMDA receptor channel block in the presence of Mg(2+) by several organic blockers with different characteristics of voltage dependence and mechanism of action. The action of NMDA receptor channel antagonists was studied in native NMDA receptors of hippocampus CA1 pyramidal neurons isolated from rat brain slices. It was demonstrated that the IC(50) values of NMDA receptor channel blockers at -30 mV are increased 1.5-5 times compared with magnesium-free conditions. The voltage dependence of the channel block is decreased, abolished or even inversed in the presence of magnesium. Although simple competition between magnesium ion and organic channel blockers provides a general explanation of the observed effects, certain disagreements were revealed. Diversity in Mg(2+) effects on the NMDAR channel block by different organic cations reported herein likely reflects interaction of NMDAR channel blockers with additional binding site(s) and suggests that individual analysis in the presence of Mg(2+) is required for newly developed NMDAR channel blocking drugs.     

Identification of ginsenoside interaction sites in 5-HT3A receptors

We previously demonstrated that 20(S)-ginsenoside Rg(3) (Rg(3)), one of the active components of Panax ginseng, non-competitively inhibits 5-HT(3A) receptor channel activity on extracellular side of the cell. Here, we sought to elucidate the molecular mechanisms underlying Rg(3)-induced 5-HT(3A) receptor regulation. We used the two-microelectrode voltage-clamp technique to investigate the effect of Rg(3) on 5-HT-mediated ion currents (I(5-HT)) in Xenopus oocytes expressing wild-type or 5-HT(3A) receptors harboring mutations in the gating pore region of transmembrane domain 2 (TM2). In oocytes expressing wild-type 5-HT(3A) receptors, Rg(3) dose-dependently inhibited peak I(5-HT) with an IC(50) of 27.6+/-4.3microM. Mutations V291A, F292A, and I295A in TM2 greatly attenuated or abolished the Rg(3)-induced inhibition of peak I(5-HT). Mutation V291A but not F292A and I295A induced constitutively active ion currents with decrease of current decay rate. Rg(3) accelerated the rate of current decay with dose-dependent manner in the presence of 5-HT. Rg(3) and TMB-8, an open channel blocker, dose-dependently inhibited constitutively active ion currents. The IC(50) values of constitutively active ion currents in V291A mutant receptor were 72.4+/-23.1 and 6.5+/-0.7microM for Rg(3) and TMB-8, respectively. Diltiazem did not prevent Rg(3)-induced inhibition of constitutively active ion currents in occlusion experiments. These results indicate that Rg(3) inhibits 5-HT(3A) receptor channel activity through interactions with residues V291, F292, and I295 in the channel gating region of TM2 and further demonstrate that Rg(3) regulates 5-HT(3A) receptor channel activity in the open state at different site(s) from those of TMB-8 and diltiazem.     

Subtype-selective antagonism of N-methyl-D-aspartate receptor ion channels by synthetic conantokin peptides

Conantokin-G (con-G), conantokin-T (con-T), a truncated conantokin-R (con-R[1-17]), that functions the same as wild-type con-R, and variant sequences of con-T, were chemically synthesized and employed to investigate their selectivities as antagonists of glutamate/glycine-evoked ion currents in human embryonic kidney-293 cells expressing various combinations of NMDA receptor (NMDAR) subunits (NR), viz., NR1a/2A, NR1a/2B, NR1b/2A and NR1b/2B. Con-G did not substantially affect ion flow into NR1a,b/NR2A-transfected cells, but potently inhibited cells expressing NR1a,b/NR2B, showing high NR2B selectivity. Con-T and con-R served as non-selective antagonists of all of four NMDAR subunit combinations. C-terminal truncation variants of the 21-residue con-T were synthesized and examined in this regard. While NMDAR ion channel antagonist activity, and the ability to adopt the Ca(2+)-induced alpha-helical conformation, diminished as a function of shortening the COOH-terminus of con-T, NMDAR subtype selectivity was enhanced in the con-T[1-11], con-T[1-9], and con-T[1-8] variants toward NR2A, NR1b, and NR1b/2A, respectively. Receptor subtype selectivity was also obtained with Met-8 sequence variants of con-T. Con-T[M8A] and con-T[M8Q] displayed selectivity with NR2B-containing subunits, while con-T[M8E] showed enhanced activity toward NR1b-containing NMDAR subtypes. Of those studied, the most highly selective variant was con-T[M8I], which showed maximal NMDAR ion channel antagonism activity toward the NR1a/2A subtype. These studies demonstrate that it is possible to engineer NMDAR subtype antagonist specificity into con-T. Since the subunit composition of the NMDAR varies temporally and spatially in developing brain and in various disease states, conantokins with high subtype selectivities are potentially valuable drugs that may be used at specific stages of disease and in selected regions of the brain.     

Ethanol sensitivity of NMDA receptor function in developing cerebellar granule neurons

The mechanism by which ethanol inhibits the function of the NMDA subtype of glutamate receptor has not been elucidated. One possibility that has been suggested is that NMDA receptor subunit composition influences the sensitivity of the receptor to ethanol. We have taken advantage of developmental changes in subunit composition of the NMDA receptor in cultured neurons to examine possible changes in the effect of ethanol. We found an increase in expression of the NR2A subunit, and a decrease in expression of the NR2B subunit of the NMDA receptor in primary cultures of cerebellar granule neurons over time in culture, with no significant change in NR1 expression. This change in NR2 subunit expression was associated with the expected changes in functional properties of the NMDA receptor (measured as the NMDA-induced increase in intracellular Ca2+), i.e., ifenprodil sensitivity and glycine potency were higher when there was a relatively greater proportion of NR2B in the cultured neurons. However, the potency of ethanol to inhibit NMDA receptor function was lower when there was a greater proportion of NR2B subunits. Previous studies showed that ethanol inhibition of NMDA receptor function in cerebellar granule neurons resulted from an ethanol-induced decrease in potency of the co-agonist, glycine, and that this effect of ethanol was blocked by inhibitors of protein kinase C. Our current results suggest that the lower potency of ethanol to inhibit the response of NMDA receptors when cerebellar granule neurons are expressing a greater proportion of NR2B subunits is a result of the higher affinity of the NMDA receptors for endogenous levels of glycine at this point in time.     

Conantokin G is an NR2B-selective competitive antagonist of N-methyl-D-aspartate receptors

Conantokin G (Con G) is a 17-amino-acid peptide antagonist of N-methyl-D-aspartate (NMDA) receptors isolated from the venom of the marine cone snail, Conus geographus. The mechanism of action of Con G has not been well defined; both competitive and noncompetitive interactions with the NMDA-binding site have been proposed. In this study the mechanism of action and subunit selectivity of Con G was examined in whole-cell voltage-clamp recordings from cultured neurons and in two electrode voltage-clamp recordings from Xenopus oocytes expressing recombinant NMDA receptors. Con G was a potent and selective antagonist of NMDA-evoked currents in murine cortical neurons (IC(50) = 480 nM). The slow onset of Con G block could be prevented by coapplication with high concentrations of NMDA or of the competitive antagonist (RS)-3-(2-carboxypiperazine-4-yl)-propyl-1-phosphonic acid. Furthermore, in oocytes expressing NR1a/NR2B receptors, Con G produced a rightward shift in the concentration-response curve for NMDA, providing support for a competitive interaction with the NMDA-binding site. Con G produced an apparent noncompetitive shift in the concentration-response curve for spermine potentiation of NMDA responses, but this was due to spermine-induced enhancement of Con G block. Spermine produced a similar enhancement of DL-2-amino-S-phosphopentanoic acid block. Finally, Con G selectively blocked NMDA receptors containing the NR2B subunit. These results demonstrate that Con G is a subunit-specific competitive antagonist of NMDA receptors. The unique subunit selectivity profile of Con G may explain its favorable in vivo profile compared with nonselective NMDA antagonists.     

Regulation of NMDA receptor function by Fyn-mediated tyrosine phosphorylation]

The ionnotropic glutamate receptor, N-methyl-D-aspartate (NMDA) receptor, is a prominent ligand-gated and voltage-gated ion channel in excitatory synaptic transmission in the mammalian central nervous system. The NMDA channel is also regulated by its phosphorylation. We have shown that an Src family kinase Fyn phosphorylates NR2A and NR2B subunits of the NMDA receptor. The phosphorylation events are facilitated by the presence of PSD-95, which is quite likely due to the complex formation of Fyn, PSD-95, and the NMDA receptor: Fyn interacts with PSD-95 and PSD-95 interacts with the NMDA receptor. We have identified tyrosine phosphorylation sites on NR2A and NR2B. A phosphorylation of one of the sites on NR2B (Tyr1472) is largely dependent on Fyn and is elevated upon the LTP induction of hippocampal CA1 neurons. The data suggest that Tyr-1472 phosphorylation of NR2B is important for synaptic plasticity. A phosphorylation of the other tyrosine residues of NR2A and NR2B would also be involved in brain development and function.     

Chronic intermittent ethanol treatment selectively alters N-methyl-D-aspartate receptor subunit surface expression in cultured cortical neurons

A chronic intermittent ethanol (CIE) exposure regimen consists of repeated episodes of ethanol intoxication and withdrawal. CIE treatment has been reported to result in a significant enhancement of N-methyl-D-aspartate (NMDA) receptor-mediated synaptic responses in vivo, and trafficking of NMDA receptors is emerging a key regulatory mechanism that underlies the channel function. Therefore, in the present study, we examined the effects of CIE on NMDA receptor subunit surface expression. Cultured cortical neurons were exposed to 75 mM ethanol for 14 h followed by 10 h of withdrawal, repeated this cycle five times, and followed by 2 or 5 days of withdrawal. Surface-expressed NMDA receptor subunits and their endocytosis were measured by biotinylation and Western blots. CIE significantly increased NMDA receptor (NR) 1 and NR2B but not NR2A subunit surface expression after 5 days of treatment. However, CIE treatment did not reduce the NMDA receptor endocytosis. Quantification of immunocytochemistry confirmed CIE-induced increase in both the total number of NR1 and NR2B subunit clusters and their targeting to synaptic sites. It is noteworthy that this effect persisted even after ethanol withdrawal with a peak expression occurring between 0 and 2 days after withdrawal, and the expression on the plasma membrane was still at high levels after 5 days of withdrawal. In addition, this was accompanied by significant increases in postsynaptic density protein 95 clusters. Protein kinase A inhibitor completely reversed CIE-induced increase in NR1 and partially in NR2B surface level and a long-lasting effect. These changes may contribute to the development of ethanol-induced neurotoxicity and ethanol dependence.     

Ethanol-related behaviors in mice lacking the NMDA receptor NR2A subunit

Rationale The ionotropic NMDA glutamate receptor is composed of NR1 and NR2 (NR2A-D) subunits. While there is compelling evidence that NMDA receptors modulate behavioral effects of ethanol, there is little understanding of how the subunit composition of the NMDA receptor mediates these effects.Objectives In the current study, we assessed the relative roles of NMDA subunits via phenotypic assessment of ethanol-related behaviors in NR2A knockout (KO) mice.Results Results demonstrated that NR2A KO and heterozygous mice failed to show evidence of ethanol-induced conditioned place preference. As compared to wild-type (WT) controls, KO mice showed impaired motor coordination at baseline and, in some instances, following ethanol treatment on the accelerating rotarod, balance beam, and wire-hang tests. By contrast, open field locomotor-stimulant, sedative/hypnotic, and hypothermic responses to ethanol were not different between genotypes, nor was voluntary ethanol consumption and preference in a two-bottle choice paradigm. Blood ethanol concentrations were lower in KO than WT mice following intraperitoneal ethanol injection.Conclusions Results suggest that the loss of NR2A subunit-containing NMDA receptors impairs the ability to form or express learned reward-related responses to ethanol and causes deficits in motor coordination. However, the loss of NR2A does not alter other measures of acute ethanol intoxication or ethanol consumption, possibly implicating other NMDA subunits in these effects. These data provide novel insight into the role of NMDA receptors in modulating the behavioral effects of ethanol.Research supported by the National Institute on Alcohol Abuse and Alcoholism Intramural Research Program.