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

Salous, AK; Ren, Huajian; Lamb, KA; Hu, Xiang‐Qun; Lipsky, Robert H.; Peoples, Robert W.

doi:10.1111/j.1476-5381.2009.00397.x

Cited by 21 publications

(18 citation statements)

References 29 publications

(76 reference statements)

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“…determined from propagated errors. Values for ethanol log IC 50 in GluN2A tryptophan mutants at Phe-637, Ala-825, and Met-823 are those reported previously (30,31,39).…”

Section: Methodssupporting

confidence: 82%

“…Electrophysiological Recording-Whole-cell patch clamp recording was performed at room temperature using an Axopatch 1D or Axopatch 200B (Axon Instruments Inc., Foster City, CA) amplifier essentially as described previously (39). Briefly, patch-pipettes had open tip resistances of 2-8 megaohms after heat polishing; series resistances of 4 -15 megaohms were compensated by 80%.…”

Section: Methodsmentioning

confidence: 99%

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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

Ren

Zhao

Dwyer

et al. 2012

Journal of Biological Chemistry

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Background:The NMDA receptor is a major target of alcohol action in the brain. Results: Four pairs of residues at the M3-M4 domain intersubunit interfaces interact to regulate gating and alcohol sensitivity. Conclusion: NMDA receptors contain putative sites of alcohol action at the M3-M4 domain interfaces. Significance: Identifying molecular sites of alcohol action on its targets is essential for understanding its actions.

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“…determined from propagated errors. Values for ethanol log IC 50 in GluN2A tryptophan mutants at Phe-637, Ala-825, and Met-823 are those reported previously (30,31,39).…”

Section: Methodssupporting

confidence: 82%

Section: Methodsmentioning

confidence: 99%

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

Ren

Zhao

Dwyer

et al. 2012

Journal of Biological Chemistry

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“…The results of the present study also did not show any relation between ethanol sensitivity and the hydrophilicity of the substituent at GluN2B(Phe637). Although such a relation was observed among mutants at GluN2A(Met823), indicating a possible role for hydrophobic binding (Ren et al , 2003b), similar relations were not observed for other positions (Ren et al , 2007; Salous et al , 2009). The lack of a clear role for molecular volume and hydrophobicity in the present study was particularly apparent when comparing the leucine and isoleucine mutants: there was a striking difference in ethanol sensitivity between the two mutants, even though they have identical physicochemical characteristics.…”

Section: Discussionmentioning

confidence: 76%

“…We and others have used alanine- and tryptophan-scanning to identify alcohol-sensitive positions in the GluN1 and GluN2A subunits (Ren et al , 2003b, 2007, 2012; Ronald et al , 2001; Salous et al , 2009; Smothers and Woodward, 2006). Previous studies in this laboratory identified several ethanol-sensitive positions in both the M3 and M4 domains of the GluN2A subunit: Phe636, Phe637, Met823, and Ala825 (Honse et al , 2004; Ren et al , 2003b, 2007, 2013).…”

Section: Discussionmentioning

confidence: 99%

Different sites of alcohol action in the NMDA receptor GluN2A and GluN2B subunits

et al. 2015

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The NMDA receptor is a major target of alcohol action in the CNS, and recent behavioral and cellular studies have pointed to the importance of the GluN2B subunit in alcohol action. We and others have previously characterized four amino acid positions in the third and fourth membrane-associated (M) domains of the NMDA receptor GluN2A subunit that influence both ion channel gating and alcohol sensitivity. In this study, we found that substitution mutations at two of the four corresponding positions in the GluN2B subunit, F637 and G826, influence ethanol sensitivity and ion channel gating. Because position 826 contains a glycine residue in the native protein, we focused our attention on GluN2B(F637). Substitution mutations at GluN2B(F637) significantly altered ethanol IC50 values, glutamate EC50 values for peak (Ip) and steady-state (Iss) current, and steady-state to peak current ratios (Iss:Ip). Changes in apparent glutamate affinity were not due to agonist trapping in desensitized states, as glutamate Iss EC50 values were not correlated with Iss:Ip values. Ethanol sensitivity was correlated with values of both Ip and Iss glutamate EC50, but not with Iss:Ip. Values of ethanol IC50, glutamate EC50, and Iss:Ip for mutants at GluN2B(F637) were highly correlated with the corresponding values for mutants at GluN2A(F636), consistent with similar functional roles of this position in both subunits. These results demonstrate that GluN2B(Phe637) regulates ethanol action and ion channel function of NMDA receptors. However, despite highly conserved M domain sequences, ethanol's actions on GluN2A and GluN2B subunits differ.

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“…Ethanol inhibition of the NMDA receptor is not determined by a single position in the membrane-associated domains, but rather the sites of ethanol action on the NMDA receptor appear to be composed of several interacting positions located in the M domains (Ren et al, 2012), each of which possess unique characteristics (Ronald et al, 2001;Ren et al, 2003bRen et al, , 2007Smothers and Woodward, 2006). For previously identified ethanol-sensitive positions in GluN2A, ethanol sensitivity was dependent on the molecular volume and hydrophilicity of the substituent at M823 (Ren et al, 2003b), was inversely dependent upon the molecular volume of the substituent at F637 (Ren et al, 2007), but was not correlated with any physicochemical measure of the substituent at A825 (Salous et al, 2009). In the GluN1 subunit, ethanol sensitivity was correlated with the molecular volume of the side chain at F639 (Smothers and Woodward, 2006), the NMDA Receptor M3 Domain Alcohol Sensitivity and Gating cognate position to GluN2A(F637).…”

Section: Discussionmentioning

confidence: 99%

A Novel Alcohol-Sensitive Position in the N-Methyl-d-Aspartate Receptor GluN2A Subunit M3 Domain Regulates Agonist Affinity and Ion Channel Gating

Ren

Zhao

et al. 2013

Mol Pharmacol

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Abundant evidence supports a role for N-methyl-D-aspartate (NMDA) receptor inhibition in the behavioral actions of ethanol, but the underlying molecular mechanisms have not been fully elucidated. We recently found that clusters of five positions in the third and fourth membrane-associated domains (M3 and M4) at the intersubunit interfaces form putative sites of alcohol action. In the present study, we found that one of these positions, NMDA receptor subunit, GluN2A(F636), can strongly regulate ethanol sensitivity, glutamate potency, and apparent desensitization: ethanol IC 50 values, peak (I p ) and steady-state (I ss ) glutamate EC 50 values, and steady-state to peak current ratio (I ss :I p ) values differed significantly among the mutants tested. Changes in glutamate affinity among the various mutants were not attributable to agonist trapping due to desensitization, as glutamate peak EC 50 values were correlated with values of both steady-state EC 50 and I ss :I p . The mean open times determined in selected mutants could be altered up to 4-fold but did not account for the changes in ethanol sensitivity. Ethanol sensitivity was significantly correlated with glutamate EC 50 and I ss :I p values, but the changes in ethanol IC 50 among mutants at this position do not appear to be secondary to changes in ion channel kinetics. Substitution of the isomeric amino acids leucine and isoleucine had markedly different effects on ethanol sensitivity, agonist potency, and desensitization, which is consistent with a stringent structural requirement for ion channel modulation by the side chain at this position. Our results indicate that GluN2A(F636) plays an important role in both channel function and ethanol inhibition in NMDA receptors.

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

Abstract: 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.

Cited by 21 publications

References 29 publications

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

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

Different sites of alcohol action in the NMDA receptor GluN2A and GluN2B subunits

A Novel Alcohol-Sensitive Position in the N-Methyl-d-Aspartate Receptor GluN2A Subunit M3 Domain Regulates Agonist Affinity and Ion Channel Gating

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