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J. Biol. Chem., Vol. 283, Issue 13, 8250-8257, March 28, 2008

This Article Full Text Full Text (PDF) All Versions of this Article: 283/13/8250    most recent M705933200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Google Scholar Articles by Ren, H. Articles by Peoples, R. W. PubMed PubMed Citation Articles by Ren, H. Articles by Peoples, R. W.

Functional Interactions of Alcohol-sensitive Sites in the N-Methyl-D-aspartate Receptor M3 and M4 Domains^*

Hong Ren, Abdelghaffar K. Salous, Jaclyn M. Paul, Kaitlin A. Lamb, Donard S. Dwyer, and Robert W. Peoples¹

From the Department of Biomedical Sciences, Marquette University, Milwaukee, Wisconsin 53201-1881 and the Department of Psychiatry, Louisiana State University Health Sciences Center, Shreveport, Louisiana 71130-3932

The N-methyl-D-aspartate receptor is an important mediator of the behavioral effects of ethanol in the central nervous system. Previous studies have demonstrated sites in the third and fourth membrane-associated (M) domains of the N-methyl-D-aspartate receptor NR2A subunit that influence alcohol sensitivity and ion channel gating. We investigated whether two of these sites, Phe-637 in M3 and Met-823 in M4, interactively regulate the ethanol sensitivity of the receptor by testing dual substitution mutants at these positions. A majority of the mutations decreased steady-state glutamate EC₅₀ values and maximal steady-state to peak current ratios (I_ss/I_p), whereas only two mutations altered peak glutamate EC₅₀ values. Steady-state glutamate EC₅₀ values were correlated with maximal glutamate I_ss/I_p values, suggesting that changes in glutamate potency were attributable to changes in desensitization. In addition, there was a significant interaction between the substituents at positions 637 and 823 with respect to glutamate potency and desensitization. IC₅₀ values for ethanol among the mutants varied over the approximate range 100–325 mM. The sites in M3 and M4 significantly interacted in regulating ethanol sensitivity, although this was apparently dependent upon the presence of methionine in position 823. Molecular dynamics simulations of the NR2A subunit revealed possible binding sites for ethanol near both positions in the M domains. Consistent with this finding, the sum of the molecular volumes of the substituents at the two positions was not correlated with ethanol IC₅₀ values. Thus, there is a functional interaction between Phe-637 and Met-823 with respect to glutamate potency, desensitization, and ethanol sensitivity, but the two positions do not appear to form a unitary site of alcohol action.

Received for publication, July 19, 2007 , and in revised form, December 13, 2007.

^* This work was supported by a grant from the Alcoholic Beverage Medical Research Foundation (R. W. P.) and National Institutes of Health Grant AA015203-01A1 (to R. W. P.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Dept. of Biomedical Sciences, SC 446, Marquette University, P.O. Box 1881, Milwaukee, WI 53201-1881. Tel.: 414-288-6678; Fax: 414-288-6564; E-mail: robert.peoples{at}marquette.edu.