Effect of the NR3 subunit on ethanol inhibition of recombinant NMDA receptors

Smothers, C. Thetford; Woodward, John J.

doi:10.1016/s0006-8993(03)03315-8

Cited by 24 publications

(23 citation statements)

References 24 publications

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“…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, 2002Matsuda et al, , 2003Smothers 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.…”

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confidence: 99%

“…The calculated reversal potential for these currents was 3.5 mV in the absence and 7.9 mV in the presence of magnesium, 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.…”

Section: Resultsmentioning

confidence: 99%

“…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).…”

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Pharmacological Characterization of Glycine-Activated Currents in HEK 293 Cells Expressing N-Methyl-D-aspartate NR1 and NR3 Subunits

Smothers

Woodward²

2007

J Pharmacol Exp Ther

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N-Methyl-D-aspartate (NMDA) receptors are important targets for drugs of abuse such as ethanol, toluene, and ketamine. Ligand-gated ion channels assembled from the NR1 and NR3 subunits have functional and pharmacological properties that are distinct from those of conventional NMDA receptors containing NR2 subunits. In the present study we used voltageclamp electrophysiology to characterize excitatory glycine-activated receptors assembled from NR1, NR3A, and NR3B subunits expressed in human embryonic kidney (HEK) 293 cells. These glycine-activated receptors were not stimulated by glutamate or kainic acid and were resistant to magnesium block. A wide variety of NMDA receptor antagonists including D-2-amino-5-phosphonovaleric acid, ifenprodil, memantine, (5R,10S)-(ϩ)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (MK-801) or acamprosate did not inhibit glycine-activated NR1/NR3A/NR3B receptors. Likewise, these receptors were not affected by antagonists of inhibitory glycine receptors or glycine transporters. The NMDA receptor glycine site agonist, D-serine, partially activated NR1/ NR3A/NR3B receptors, whereas the antagonist, 5,7-dichlorokynurenic acid, inhibited receptor currents. Conversely, the antagonist, 7-chlorokynurenic acid, and the partial agonist, R-(ϩ)-3-amino-1-hydroxy-2-pyrrolidinone (HA-966), potentiated glycine-stimulated currents of these receptors. NR1/ NR3A/NR3B receptor currents were inhibited by 10 to 21% by ethanol and toluene but were relatively insensitive to ketamine. Ethanol inhibition was enhanced in receptors expressing the NR1(L819A) mutant, whereas those containing NR1(F639A) or NR1(M813A) showed no change relative to the wild-type NR1. The results of this study indicate that coexpression of NR1, NR3A, and NR3B subunits in HEK 293 cells results in glycineactivated receptors with novel functional and pharmacological properties.N-Methyl-D-aspartate (NMDA) receptors are glutamateactivated ion channels that contain NR1 and NR2 subunits. A third class of NMDA receptor subunits has also been reported and consists of the NR3A and NR3B subunits (Ciabarra et al., 1995;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...

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confidence: 99%

Section: Resultsmentioning

confidence: 99%

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confidence: 99%

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Pharmacological Characterization of Glycine-Activated Currents in HEK 293 Cells Expressing N-Methyl-D-aspartate NR1 and NR3 Subunits

Smothers

Woodward²

2007

J Pharmacol Exp Ther

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“…Moreover, NMDARs with NR1/NR2B subunit combination were more susceptible to the effect of ethanol compared to those composed of NR1/NR2A subunits [7,14,15,192]. The co-expression of NR3 or an NR3-GFP fusion protein with NR1/NR2 (A-D) subunits did not alter the inhibitory effects of ethanol [193].…”

Section: Ethanol Is a Potent Inhibitor Of Nmda Receptorsmentioning

confidence: 89%

Role of Altered Structure and Function of NMDA Receptors in Development of Alcohol Dependence

Nagy

Kolok²,

Boros³

et al. 2005

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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.

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“…Thus, enhancing gating parameters via A7 mutations might be expected to generate receptors that are less sensitive to ethanol-induced changes in channel function. It should be noted that although the actions of various allosteric modulators including zinc, protons, and ifenprodil require the amino-terminal domain of the receptor, ethanol inhibition is unaltered in NMDA receptors lacking this domain (Smothers et al, 2009).…”

Section: Downloaded Frommentioning

confidence: 99%

Ethanol Inhibition of Constitutively Open N-Methyl-d-Aspartate Receptors

Smothers²,

Trudell³

et al. 2011

J Pharmacol Exp Ther

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N-Methyl-D-aspartate (NMDA) receptors gate a slow and calciumrich component of the postsynaptic glutamate response. Like all ionotropic glutamate receptors, NMDA subunits contain a highly conserved motif (SYTANLAAF) in the transmembrane (TM) 3 domain that is critically involved in channel gating. Mutation of an alanine in this domain (A7; underlined above) results in constitutively open receptors that show reduced sensitivity to several allosteric modulators. In this study, we examined the effects of ethanol, a substance that inhibits NMDA currents via an unknown mechanism, on tonically active NMDA receptors expressed in human embryonic kidney 293 cells. Ethanol (100 mM) inhibited currents from GluN1(A7R)/GluN2A and GluN1(A7R)/GluN2B receptors by approximately 50%, whereas those from GluN1/ GluN2B(A7R) receptors were reduced by less than 10%. In cysteine-substituted GluN1 and GluN2 A7 mutants, estimated ethanol IC 50 values for agonist-gated currents were 101, 117, 103, and 69 mM for GluN1(A7C)/GluN2A, GluN1(A7C)/GluN2B, GluN1/ GluN2A(A7C), and GluN1/GluN2B(A7C) receptors, respectively. After exposure to the thiol-modifying reagent 2-(trimethylammonium)ethyl methanethiosulfonate (MTSET), A7C mutants showed robust agonist-independent currents and reduced sensitivity to ethanol (IC 50 values of 371, 256, 715, and 958 mM, respectively, as above). In contrast, cysteine modification of the ligand-binding domain resulted in constitutively open receptors that showed robust ethanol inhibition. Ethanol inhibition of MTSET-treated GluN1(A7C) receptors was further reduced by TM3/TM4 mutations previously shown to reduce ethanol sensitivity of agonistgated receptors. Overall, these results show that ethanol affects NMDA receptor function at a site distal from agonist binding and appears to exert greater effects via perturbation of GluN2 subunits.

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Effect of the NR3 subunit on ethanol inhibition of recombinant NMDA receptors

Cited by 24 publications

References 24 publications

Pharmacological Characterization of Glycine-Activated Currents in HEK 293 Cells Expressing N-Methyl-D-aspartate NR1 and NR3 Subunits

Pharmacological Characterization of Glycine-Activated Currents in HEK 293 Cells Expressing N-Methyl-D-aspartate NR1 and NR3 Subunits

Role of Altered Structure and Function of NMDA Receptors in Development of Alcohol Dependence

Ethanol Inhibition of Constitutively Open N-Methyl-d-Aspartate Receptors

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