![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
TOXICOLOGY
Department of Neurosciences, Division of Neuroscience Research, and Center for Drug and Alcohol Programs, Medical University of South Carolina, Charleston, South Carolina
Received for publication
April 2, 2007
Accepted
May 11, 2007.
 |
Abstract |
|---|
 Top Abstract Materials and MethodsResultsDiscussionReferences |
|---|
; Sun et al., 1998). NR3 subunits share structural features with the NR1 subunit, including the S1 and S2 agonist binding domains and some sequence similarity across transmembrane domains (Chatterton et al., 2002). Coexpression of the NR3A or NR3B subunits with NR1 and NR2 in recombinant expression systems decreases NMDA receptor current (Chatterton et al., 2002) and reduces calcium permeability (Matsuda et al., 2002). In NR3A-deficient mice, NMDA receptor currents are significantly enhanced (Das et al., 1998), suggesting that these subunits may be important modulators of NMDA receptor function.
As for NR1 subunits, NR3 subunits do not form functional channels when expressed alone. However, coexpression of NR1 and NR3 subunits in oocytes has been reported to produce functional channels that are gated by glycine but not glutamate (Chatterton et al., 2002; but see Smothers and Woodward, 2003). In oocytes, NR1/NR3 receptors display novel pharmacological and functional properties, and the NR1 subunit seems to markedly influence the function of NR1/NR3 receptors (Wada et al., 2006; Awobuluyi et al., 2007; Madry et al., 2007). Previous studies from our laboratory have also shown that the NR1 subunit modulates the ethanol sensitivity of NMDA receptors (Ronald et al., 2001; Jin and Woodward, 2006; Smothers and Woodward, 2006).
The goal of the present study was to determine whether glycine-activated NR1/NR3 receptors are also sensitive to drugs of abuse such as ethanol. In contrast to studies reporting glycine-induced currents in oocytes expressing NR1/NR3 dimers (Chatterton et al., 2002; Wada et al., 2006; Awobuluyi et al., 2007; Madry et al., 2007), transfection of these subunits into mammalian cells does not produce functional receptors (Nishi et al., 2001; Matsuda et al., 2002, 2003; Smothers and Woodward, 2003). However, as reported in the present study, simultaneous transfection of cDNAs encoding NR1, NR3A, and NR3B subunits into HEK 293 cells produces robust glycine-activated currents. In the present study, we determined the functional characteristics of these channels as well as their sensitivity to ethanol and other drugs of abuse.
|
Materials and Methods |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
).
Maintenance of HEK 293 Cells and Recombinant Receptor Expression. HEK 293 cells (American Type Culture Collection, Manassas, VA) were grown and maintained as described previously (Smothers and Woodward, 2003). In brief, cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal calf serum (Hyclone, Logan, UT) and grown at 37°C in a 5% CO2 environment. Twenty-four hours after plating of low-density cultures (approximately 5 x 104 cells per dish) onto 35-mm dishes, cells were transfected with equal amounts of cDNA (1 µg) coding for NR1 (wild-type or mutant), NR3A, and NR3B using the Lipofectamine 2000 reagent (Invitrogen). Cells were used for electrophysiological recordings 24 to 48 h after transfection.
Electrophysiological Recording Conditions. All recordings were performed as described previously (Smothers and Woodward, 2006). In brief, cells were perfused with an external solution containing 135 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2, 5 mM HEPES, and 10 mM glucose (pH adjusted to 7.2 with NaOH and osmolarity adjusted to 290–300 mOsmol/kg with sucrose). The pipette filling solution was composed of 100 mM N-methyl-D-glucamine, 40 mM CsCl, 2 mM NaATP, 2 mM MgCl2, 10 mM HEPES, and 10 mM EGTA (pH was adjusted to 7.2 with KOH and osmolarity was adjusted to 325 mOsmol/kg with sucrose). In experiments examining current-voltage relationships, the internal solution contained 140 mM KCl, 1 mM CaCl2, 4 mM NaATP, 6 mM MgCl2, 10 mM HEPES, and 5 mM EGTA (pH was adjusted to 7.2 with KOH and osmolarity was adjusted to 290–300 mOsmol/kg with sucrose). All internal solutions used for each experiment were from frozen stocks. All drug solutions were prepared fresh for each experiment from frozen stocks in external solution. Stock solutions of glycine, glutamate, kainic acid, L-alanine, D-serine, APV, memantine, ifenprodil, MK-801, strychnine, ketamine, and HA-966 were prepared in water and diluted into external solution. Stock solutions of cyclothiazide, 7-chlorokynurenic acid (7-CK), 5,7-dichlorokynurenic acid (5,7-DCK), N-[3-(4'fluorophenyl)-3–4'-phenylphenoxy)propyl]sarcosine (NFPS), and O-[(2-benzyloxyphenyl-3-flurophenyl)methyl]-L-serine (ALX-1393) were prepared in dimethylsulfoxide. The final concentration of DMSO used in these experiments did not exceed 1.0%, and no effect of vehicle was observed on current traces (data not shown). Ethanol was purchased from Aaper Alcohol and Chemical (Shelbyville, KY). DL-threo-
-Benzyloxyaspartate was purchased from Tocris Bioscience (Ellisville, MO). All other chemicals were purchased from Sigma-Aldrich (St. Louis, MO).
Whole-cell voltage-clamp recordings (Hamill et al., 1981) were performed at room temperature using the Axopatch 200B patch-clamp amplifier (Molecular Devices, Sunnyvale, CA). Cells were voltage-clamped at –55 or –60 mV, and current records were filtered at 1 kHz (eight-pole Bessel filter) and digitized at 2 kHz using an ITC-16 interface (InstruTECH Corporation, Port Washington, NY). Software control of data acquisition was provided by Pulse Control running within the Igor Pro program (version 4.03; Wave-Metrics, Portland, OR) on an Apple Macintosh G3 computer (Apple Computer, Cupertino, CA). Patch electrodes were fabricated from thick-walled borosilicate glass (B150; WPI, Sarasota, FL) and filled with internal solution (tip resistance 4–7 M
). A three-barrel perfusion apparatus (barrel i.d. 0.6 mm, SF-77B; Warner Instruments, Hamden, CT) was used to switch between control and drug-containing solutions. Solution exchange times were determined to be between 6 and 8 ms as calculated by measuring changes in the liquid junction current across an open electrode when switching between solutions with different ionic strength.
Currents evoked by a 6-s agonist application were measured at two time points, peak and steady-state. Peak current amplitude was determined as the difference between the current immediately before agonist application and at the point where inward current was maximal. Steady-state current amplitude was determined as the difference between current immediately before agonist application and that 4.5 s into the agonist application at which currents were stable. Drug-induced inhibition of receptor currents (IControl) was calculated using the formula [1–(IGlycine+drug/IControl)] x 100, where IGlycine+drug represents the response to coapplication of glycine + drug, and IControl represents the mean of two responses to glycine, one before and one after the coapplication of the drug. Leak currents were continually monitored as an indicator of seal and cell integrity. Cells that showed unstable leak currents were not included in the data analysis.
Data Analysis. Data were analyzed by analysis of variance (ANOVA) and post hoc testing using Prism 4.0 (GraphPad Software Inc., San Diego, CA). All drug inhibition data are expressed as the percentage of the average control response (mean ± S.E.M.) unless otherwise stated, where n represents the number of cells. Curve fitting for dose-response curves was performed using the following equation: Y = Ymin + (Ymax–Ymin)/(1 + 10[log10(EC50)–X]), where X is log10[drug], and Y is percentage of maximal current.
|
Results |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
|
Classic NMDA receptors contain NR1 and NR2 subunits and require glutamate and glycine for activation with NR1 subunits supplying the binding site for glycine and NR2 subunits providing the site for glutamate binding (Dingledine et al., 1999). In HEK cells transfected with NR1, NR2, and NR3 subunits, coapplication of glutamate and glycine induced inward currents that showed little desensitization. No currents were generated when these agonists were applied separately (Fig. 2A), suggesting that glycine-activated receptors are not formed in the presence of NR2 subunits. As for NR1 subunits, NR3 subunits also contain a high-affinity binding site for glycine (Yao and Mayer, 2006). To determine whether this binding site could support functional receptors in combination with NR2 subunits, HEK 293 cells were transfected with the NR3 subunits and various NR2 subunits (NR2A, NR2B, NR2C, and NR2D). Under these conditions, only relatively small (<30 pA) currents were evoked during agonist application (Fig. 2, B and C), suggesting that NR2/NR3 subunits do not form functionally relevantion channels.
|
To further characterize the functional profile of NR3-containing receptors, a variety of agents with actions on glycine and NMDA receptors were tested on cells expressing NR1/NR3A/NR3B receptors.
Effects of Glycine Transporter Antagonists. As shown in Fig. 3A, glycine-induced currents were not affected by NFPS, an antagonist at the glial localized glycine transporter-1 (GLYT1). Likewise, ALX-1393 (1–10 µM), an antagonist at the neuronal localized glycine transporter-2 (GLYT2), did not alter currents evoked by glycine (Fig. 3A; n = 7 cells)
|
-alanine, and taurine, and the antagonist, strychnine, were tested. The application of L-alanine produced either no current or small inward currents (<50 pA; n = 7 cells), and/or no currents were observed during exposure to either -alanine or taurine (Fig. 3B; n = 4 cells). In addition, acampro-sate (100 µM), a taurine derivative that is currently used in the treatment of alcohol dependence (Rammes et al., 2001), did not induce or alter glycine-induced currents from NR1/NR3A/NR3B receptors (n = 3 cells, data not shown). In addition, neither the glycine receptor antagonist, strychnine (Fig. 3B; n = 7 cells), or the antagonist at the GABAA antagonist, picrotoxin (Fig. 3B; n = 3 cells), had any effect on glycine-induced currents in cells transfected with NR1/NR3A/NR3B subunits. Effects of NMDA Partial Agonists. A series of experiments were conducted using a variety of compounds that show selectivity for NMDA receptors. D-Serine, an agonist at the glycine site of the NMDA receptor, reduced the amplitude of glycine-evoked currents from NR1/NR3A/NR3B transfected cells suggesting that D-serine is a partial agonist at these receptors. This was verified in experiments in which the responses of the same cell to both glycine and D-serine was measured. As shown in Fig. 4A, both glycine and D-serine induced responses from NR1/NR3A/NR3B transfected cells (n = 5). However, the maximal peak current produced by D-serine was approximately 25% of that produced by 100 µM glycine (Fig. 4B).
|
). HA-966 had no effect on currents generated in cells expressing the NR3 subunits and the NR1(D481N) mutant (Fig. 5D; n = 9). A similar lack of effect of HA-966 was observed in cells expressing NR3 subunits and the NR1(D732N) mutant (Fig. 5E; n = 6). As shown in Fig. 5F, currents were enhanced with glycine but not with D-serine or in mutants with decreased glycine sensitivity.
|
|
|
Effects of Ethanol. Because NMDA receptors composed of NR1 and NR2 subunits are inhibited by ethanol (Smothers and Woodward, 2003), we tested the effects of ethanol on NR1/NR3A/NR3B receptors. As shown in Fig. 8, ethanol (100 mM) inhibited peak and steady-state glycine-activated currents from NR1/NR3A/NR3B transfected cells by 8 and 21%, respectively. The inhibition was concentration-dependant with 50 mM ethanol inhibiting the steady-state current by 14.9 ± 4.7%, 25 mM by 4.7 ± 1.7%, and 10 mM by 2.4 ± 13.3% (n = 4 cells per concentration). Ethanol inhibition of glycine-activated currents was also determined in cells transfected with mutant NR1 subunits shown to either reduce (F639A) or enhance (M813A and L819A) the ethanol sensitivity of NR1/NR2 receptors (Ronald et al., 2001; Smothers and Woodward, 2006). Cells transfected with NR3A/NR3B subunits and NR1(F639A), NR1(M813A), or NR1(L819A) subunits were functional (Fig. 8, B–D). However, currents from NR1(L819A)-containing receptors were consistently smaller than those from other mutant or wild-type receptors. In contrast to wild-type receptors, neither the NR1(F639A) or NR1(M813A) mutant altered ethanol inhibition of NR1/NR3A/NR3B receptors (Fig. 8E). In contrast, ethanol inhibition was enhanced in receptors containing the NR1(L819A) mutant compared with wild-type NR1/NR3A/NR3B receptors [ANOVA (p < 0.02) with unpaired t test (p < 0.01)] (Fig. 8E).
|
). Previous studies from our laboratory have shown that both recombinant and native NMDA receptors are inhibited by toluene, with NR1/NR2B receptors being nearly completed inhibited by concentrations of 1 mM and higher (Cruz et al., 1998; Bale et al., 2005). In the present study, we extended these findings and tested whether NR1/NR3A/NR3B receptors are also sensitive to modulation by toluene. Toluene (1 mM) had only modest effects on glycine-activated responses and inhibited peak and steady-state currents by 4.7 ± 4.3 and 13.29 ± 7.94%, respectively (Fig. 9, A and C).
|
). Ketamine blocks NMDA receptors through an interaction with sites thought to be located within the ion channel pore region (Orser et al., 1997). Recently, it was shown that inclusion of the NR3B subunit does not alter the ketamine sensitivity of recombinant NR1/NR2 receptors expressed in oocytes (Yamakura et al., 2005), Likewise, 100 µM ketamine produced only weak inhibition (4.3 ± 3.3% peak component and 7.9 ± 6.8% steady-state component) of the glycine-induced current of NR1/NR3A/NR3B receptors (Fig. 9, B and C).
|
Discussion |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
).
In previous studies, glycine has been shown to evoke large inward currents in oocytes injected with mRNA coding for either NR1/NR3A or NR1/NR3B subunits (Chatterton et al., 2002; Madry et al., 2007). However, in the present study and in others, these responses are not observed in mammalian cells transfected with NR1/NR3A or NR1/NR3B subunits (Nishi et al., 2001; Matsuda et al., 2002, 2003). This lack of consistent findings between expression systems could be the result of differences in receptor trafficking or to the presence of endogenous NMDA-like subunits in Xenopus oocytes (Soloviev et al., 1996). In the present study, glycine-activated currents in HEK 293 cells was dependent upon expression of three subunits, NR1, NR3A, and NR3B. The NR1 subunit seems to be critical as no current was produced when it was absent or substituted with an NR2 subunit. Although NR3 subunits also bind glycine as do NR1 subunits, no glutamate/glycine-activated currents were induced when the NR3 was paired with any NR2 subunit. Furthermore, expressing NR3 subunits in combination with NR1 and NR2 subunits yields receptors that require both glutamate and glycine suggesting that glycine-activated NR1/NR3A/NR3B receptors may not exist in great abundance in the presence of NR2 subunits.
Although no functional currents were evident with dimeric NR1/NR3 subunit combinations in HEK 293 cells, currents from cells transfected with NR1/NR3A/NR3B subunits are functionally and pharmacologically similar to NR1/NR3 receptors expressed in oocytes (Chatterton et al., 2002; Wada et al., 2006; Awobuluyi et al., 2007; Madry et al., 2007). Consistent with findings of Chatterton et al. (2002), the most efficacious agonist to activate the receptors was glycine followed by D-serine, which at NR1/NR3A/NR3B receptors is a weak partial agonist. In addition, similar findings were reported for the NMDA receptor antagonists, APV, MK-801, memantine, and ifenprodil, that inhibit NR1/NR2 receptors but do not inhibit glycine-activated currents from NR1/NR3A/NR3B receptors. The inability of APV and ifenprodil to inhibit NR3 receptors is consistent with a lack of NR2-specific binding sites for these compounds (Williams, 1993; Clements and Westbrook, 1994). The inability of the channel blockers MK-801, ketamine, and memantine (Huettner and Bean, 1988; Chen and Lipton, 1997) to block NR1/NR3A/NR3B receptors suggests that the channel pore domain of these receptors is different from that of classic NR1/NR2 NMDA receptors. This finding is consistent with the lack of magnesium sensitivity of NR1/NR3A/NR3B receptors as shown by the linear current-voltage relationship. These findings agree well with other reports in which NR3 receptor expression in oocytes was studied and indicate that excitatory glycine-activated receptors formed from NMDA subunits are a distinct pharmacological entity from classic NR2-containing NMDA receptors.
The NMDA glycine site antagonists, 7-CK and 5,7-DCK, inhibited glycine-activated currents of NR1/NR3A/NR3B receptors. However, the inhibition by 7-CK was complex. Peak and steady-state components of the current were inhibited at low concentrations, whereas at higher concentrations the peak component was completely blocked, but the steady-state component was potentiated. The most parsimonious explanation for this finding is that activation of the NR1 subunit negatively influences channel opening and that inhibiting this interaction results in current potentiation. This finding is consistent with results from recent studies of NR3 receptors expressed in oocytes (Awobuluyi et al., 2007; Madry et al., 2007). In those studies, blocking or reducing glycine binding to the NR1 subunit markedly attenuated the desensitizing component observed during application of high concentrations of glycine and greatly enhanced steady-state currents. In contrast to 7-CK, 5,7-DCK inhibited both the peak and steady-state components of the current and did not potentiate steady-state currents. This finding is different from that reported by Awobuluyi et al. (2007) who showed that 5,7-DCK could also potentiate the glycine-activated current. The difference in antagonist action could be due to subtle alterations in receptor subunit composition given that trimeric NR1/NR3A/NR3B receptors are used in this study versus dimeric NR3 receptors in the Awobuluyi et al. (2007) study. Overall differences between 7-CK and 5,7-DCK on NR3 receptor currents could be due to differences in the sites of action for these drugs (Baron et al., 1990, 1991).
Unlike results obtained with glycine site antagonists, the NMDA receptor partial agonist, HA-966, only potentiated glycine-activated currents with both peak and steady-state components being affected. The potentiation by HA-966 is similar to that reported for submicromolar concentrations of the glycine antagonist MDL-29951 in a study of dimeric NR1/NR3 receptors expressed in oocytes (Madry et al., 2007). The ability of HA-966 and MDL-29951 to potentiate glycine-induced currents suggests that these compounds interact specifically with the NR1 glycine site to reduce glycine-induced desensitization of NR3-containing receptors.
NMDA receptors have been suggested to be important targets for drugs of abuse, including ethanol (Lovinger et al., 1989; Peoples et al., 1997), ketamine (Petrovi
et al., 2005), and abused inhalants, such as toluene (Cruz et al., 1998, 2000). In the present study, we found that NR1/NR3A/NR3B receptors were inhibited to some degree by ethanol, toluene, and ketamine although this inhibition varied depending on the drug. The greatest degree of inhibition was observed with ethanol (20% decrease) followed by toluene (15%) and ketamine (5%). At the concentrations tested, both toluene and ketamine almost completely blocked currents generated by specific members of the classic NR1/NR2 receptor family (Cruz et al., 1998; Ogata et al., 2006). This finding suggests that these drugs may selectively target residues within NR2 rather than NR1 subunits to cause their effects. The situation with ethanol is less clear because the difference in the degree of inhibition of NR2- and NR3-containing receptors by ethanol is not as great. In addition, the ethanol inhibition of NR3-containing receptors was not consistently modulated by mutagenesis of the NR1 subunit. Previous studies have identified NR1 mutants that enhance (M813A and L819A) or reduce (F639A) the ethanol sensitivity of NR1/NR2 NMDA receptors (Ronald et al., 2001; Smothers and Woodward, 2006). However, in NR3-containing receptors, only the NR1(L819A) mutant was effective in altering the ethanol inhibition of the receptor. These findings suggest that these amino acids may play different roles in the activation and regulation of NMDA receptor activity between NR2- and NR3-containing receptors.
In conclusion, this study demonstrates excitatory glycine-activated receptors in a mammalian expression system. These receptors have novel functional and pharmacological properties and are sensitive to drugs of abuse. Given the findings of this study, NR3 receptors constitute new targets for the actions of ethanol and toluene.
|
Footnotes |
|---|
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: NMDA, N-methyl-D-aspartate; HEK, human embryonic kidney; APV, D-2-amino-5-phosphonovaleric acid; MK-801, (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate; HA-966, R-(+)-3-amino-1-hydroxy-2-pyrrolidinone; 7-CK, 7-chlorokynurenic acid; 5,7-DCK, 5,7-dichlorokynurenic acid; NFPS, N-[3-(4'fluorophenyl)-3–4'-phenylphenoxy)propyl]sarcosine; ALX-1393, O-[(2-benzyloxyphenyl-3-flurophenyl)methyl]-L-serine; ANOVA, analysis of variance; MDL-29951, 3-(4,6-dichloro-2-carboxymethylamino-5,7-dichloroquinoline-2-carboxylic acid.
Address correspondence to: Dr. John J. Woodward, MUSC CDAP IOP 4 North, Box 250861, Charleston, SC 29425. E-mail: woodward{at}musc.edu
|
References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Awobuluyi M, Yang J, Ye Y, Chatterton JE, Godzik A, Lipton SA, and Zhang D (2007) Subunit-specific roles of glycine-binding domains in activation of NR1/NR3 "NMDA" receptors. Mol Pharmacol 71: 112–122.
Bale AS, Meacham CA, Benignus VA, Bushnell PJ, and Shafer TJ (2005) Volatile organic compounds inhibit human and rat neuronal nicotinic acetylcholine receptors expressed in Xenopus oocytes. Toxicol Appl Pharmacol 205: 77–88.[CrossRef][Medline]
Baron BM, Harrison BL, Miller FP, McDonald IA, Salituro FG, Schmidt CJ, Sorensen SM, White HS, and Palfreyman MG (1990) Activity of 5,7-dichlorokynurenic acid, a potent antagonist at the N-methyl-D-aspartate receptor-associated glycine binding site. Mol Pharmacol 38: 554–561.[Abstract]
Baron BM, Siegel BW, Slone AL, Harrison BL, Palfreyman MG, and Hurt SD (1991) [3H]5,7-Dichlorokynurenic acid, a novel radioligand labels NMDA receptor-associated glycine binding sites. Eur J Pharmacol 206: 149–154.[CrossRef][Medline]
Betz H and Laube B (2006) Glycine receptors: recent insights into their structural organization and functional diversity. J Neurochem 97: 1600–1610.[CrossRef][Medline]
Chatterton JE, Awobuluyi M, Premkumar LS, Takahashi H, Talantova M, Shin Y, Cui J, Tu S, Sevarino KA, Nakanishi N, Tong G, Lipton SA, and Zhang D(2002) Excitatory glycine receptors containing the NR3 family of NMDA receptor subunits. Nature 415: 793–798.[Medline]
Chen HS and Lipton SA (1997) Mechanism of memantine block of NMDA-activated channels in rat retinal ganglion cells: uncompetitive antagonism. J Physiol 499: 27–46.
Ciabarra AM, Sullivan JM, Gahn LG, Pecht G, Heinemann S, and Sevarino KA (1995) Cloning and characterization of 
-1: A developmentally regulated member of a novel class of the ionotropic glutamate receptor family. J Neurosci 15: 6498–6508.
Clements JD and Westbrook GL (1994) Kinetics of AP5 dissociation from NMDA receptors: evidence for two identical cooperative binding sites. J Neurophysiol 71: 2566–2569.
Cruz SL, Balster RL, and Woodward JJ (2000) Effects of volatile solvents on recombinant N-methyl-D-aspartate receptors expressed in Xenopus oocytes. Br J Pharmacol 131: 1303–1308.[CrossRef][Medline]
Cruz SL, Mirshahi T, Thomas B, Balster RL, and Woodward JJ (1998) Effects of the abused solvent toluene on recombinant N-methyl-D-aspartate and non-N-methyl-D-aspartate receptors expressed in Xenopus oocytes. J Pharmacol Exp Ther 286: 334–340.
Das S, Sasaki YF, Rothe T, Premkumar LS, Takasu M, Crandall JE, Dikkes P, Conner DA, Rayudu PV, Cheung W, et al. (1998) Increased NMDA current and spine density in mice lacking the NMDA receptor subunit NR3A. Nature 393: 377–381.[CrossRef][Medline]
Dingledine R, Borges K, Bowie D, and Traynelis SF (1999) The glutamate receptor ion channels. Pharmacol Rev 51: 7–61.
Freese TE, Miotto K, and Reback CJ (2002) The effects and consequences of selected club drugs. J Subst Abuse Treat 23: 151–156.[CrossRef][Medline]
Hamill OP, Marty A, Neher E, Sakmann B, and Sigworth FJ (1981) Improved patch-clamp techniques for high-resolution current recording from cells and cellfree membrane patches. Pflugers Arch 391: 85–100.[CrossRef][Medline]
Huettner JE and Bean BP (1988) Block of N-methyl-D-aspartate-activated current by the anticonvulsant MK-801: selective binding to open channels. Proc Natl Acad Sci USA 85: 1307–1311.
Jin C and Woodward JJ (2006) Effects of 8 different NR1 splice variants on the ethanol inhibition of recombinant NMDA receptors. Alcohol Clin Exp Res 30: 673–679.[CrossRef][Medline]
Kurtzman TL, Otsuka KN, and Wahl RA (2001) Inhalant abuse by adolescents. J Adolesc Health 28: 170–180.[CrossRef][Medline]
Lovinger DM, White G, and Weight FF (1989) Ethanol inhibits NMDA-activated ion current in hippocampal neurons. Science 243: 1721–1724.
Madry C, Mesic I, Bartholomaus I, Nicke A, Betz H, and Laube B (2007) Principal role of NR3 subunits in NR1/NR3 excitatory glycine receptor function. Biochem Biophys Res Commun 354: 102–108.[CrossRef][Medline]
Matsuda K, Fletcher M, Kamiya Y, and Yuzaki M (2003) Specific assembly with the NMDA receptor 3B subunit controls surface expression and calcium permeability of NMDA receptors. J Neurosci 23: 10064–10073.
Matsuda K, Kamiya Y, Matsuda S, and Yuzaki M (2002) Cloning and characterization of a novel NMDA receptor subunit NR3B: a dominant subunit that reduces calcium permeability. Brain Res Mol Brain Res 100: 43–52.[Medline]
Nishi M, Hinds H, Lu HP, Kawata M, and Hayashi Y (2001) Motoneuron-specific expression of NR3B, a novel NMDA-type glutamate receptor subunit that works in a dominant-negative manner. J Neurosci 21: RC 185.
Ogata J, Shiraishi M, Namba T, Smothers CT, Woodward JJ, and Harris RA (2006) Effects of anesthetics on mutant N-methyl-D-aspartate receptors expressed in Xenopus oocytes. J Pharmacol Exp Ther 318: 434–443.
Orser BA, Pennefather PS, and MacDonald JF (1997) Multiple mechanisms of ketamine blockade of N-methyl-D-aspartate receptors. Anesthesiology 86: 903–917.[CrossRef][Medline]
Peoples RW, White G, Lovinger DM, and Weight FF (1997) Ethanol inhibition of N-methyl-D-aspartate-activated current in mouse hippocampal neurones: wholecell patch-clamp analysis. Br J Pharmacol 122: 1035–1042.[CrossRef][Medline]
Petrovi M, Horak M, Sedlacek M and Vyklicky L Jr (2005) Physiology and pathology of NMDA receptors. Prague Med Rep 106: 113–136.[Medline]
Rammes G, Mahal B, Putzke J, Parsons C, Spielmanns P, Pestel E, Spanagel R, Zieglgansberger W, and Schadrack J (2001) The anti-craving compound acamprosate acts as a weak NMDA-receptor antagonist, but modulates NMDA-receptor subunit expression similar to memantine and MK-801. Neuropharmacology 40: 749–760.[CrossRef][Medline]
Ronald KM, Mirshahi T, and Woodward JJ (2001) Ethanol inhibition of N-methyl-D-aspartate receptors is reduced by site-directed mutagenesis of a transmembrane domain phenylalanine residue. J Biol Chem 276: 44729–44735.
Smothers CT and Woodward JJ (2003) Effect of the NR3 subunit on ethanol inhibition of recombinant NMDA receptors. Brain Res 987: 117–121.[CrossRef][Medline]
Smothers CT and Woodward JJ (2006) Effects of amino acid substitutions in transmembrane domains of the NR1 subunit on the ethanol inhibition of recombinant N-methyl-D-aspartate receptors. Alcohol Clin Exp Res 30: 523–530.[CrossRef][Medline]
Soloviev MM, Brierley MJ, Shao ZY, Mellor IR, Volkova TM, Kamboj R, Ishimaru H, Sudan H, Harris J, Foldes RL, et al. (1996) Functional expression of a recombinant unitary glutamate receptor from Xenopus, which contains N-methyl-D-aspartate (NMDA) and non-NMDA receptor subunits. J Biol Chem 271: 32572–32579.
Sun LX, Margolis FL, Shipley MT, and Lidow MS (1998) Identification of a long variant of mRNA encoding the NR3 subunit of the NMDA receptor: its regional distribution and developmental expression in the rat brain. FEBS Lett 441: 392–396.[CrossRef][Medline]
Wada A, Takahashi H, Lipton SA, and Chen HS (2006) NR3A modulates the outer vestibule of the "NMDA" receptor channel. J Neurosci 26: 13156–13166.
Wafford KA, Kathoria M, Bain CJ, Marshall G, Le Bourdellès B, Kemp JA, and Whiting PJ (1995) Identification of amino acids in the N-methyl-D-aspartate receptor NR1 subunit that contribute to the glycine binding site. Mol Pharmacol 47: 374–380.[Abstract]
Williams K (1993) Ifenprodil discriminates subtypes of the N-methyl-D-aspartate receptor: selectivity and mechanisms at recombinant heteromeric receptors. Mol Pharmacol 44: 851–859.[Abstract]
Yamakura T, Askalany AR, Petrenko AB, Kohno T, Baba H, and Sakimura K (2005) The NR3B subunit does not alter the anesthetic sensitivities of recombinant N-methyl-D-aspartate receptors. Anesth Analg 100: 1687–1692.
Yao Y and Mayer ML (2006) Characterization of a soluble ligand binding domain of the NMDA receptor regulatory subunit NR3A. J Neurosci 26: 4559–4566.
This article has been cited by other articles:
|
|
 |
|
  M. H. Ulbrich and E. Y. Isacoff Rules of engagement for NMDA receptor subunits PNAS, September 16, 2008; 105(37): 14163 - 14168. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Tong, H. Takahashi, S. Tu, Y. Shin, M. Talantova, W. Zago, P. Xia, Z. Nie, T. Goetz, D. Zhang, et al. Modulation of NMDA Receptor Properties and Synaptic Transmission by the NR3A Subunit in Mouse Hippocampal and Cerebrocortical Neurons J Neurophysiol, January 1, 2008; 99(1): 122 - 132. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|
 |
 |
 |
) and 5,7-DCK (
). Data represent the percent change of steady-state currents by each drug and are expressed as the means ± S.E.M (n = 3–5 cells).
