Ethanol Inhibition ofN-Methyl-d-aspartate Receptors Is Reduced by Site-directed Mutagenesis of a Transmembrane Domain Phenylalanine Residue

Kimberly M Ronald,Tooraj Mirshahi,John J Woodward

doi:10.1074/jbc.m102800200

Kimberly M Ronald, Tooraj Mirshahi + Show 1 more

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https://doi.org/10.1074/jbc.m102800200

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Abstract

N-Methyl-D-aspartate (NMDA) receptors (NRs) are ionotropic receptors activated by glutamate and the co-agonist glycine. Ethanol inhibits NMDA receptor function, although its site of action is undefined. We hypothesized that ethanol acts at specific amino acids contained within the transmembrane (TM) domains of the receptor. In this study, NR1 and NR2A subunits were altered by mutagenesis and tested for sensitivity to ethanol. Three NR1 mutants (W636A, F817A, and L819A) and one NR2A mutant (F637A) failed to generate functional receptors. Pre-TM1 (I546A, L551A, F554A, and F558A), TM1 (W563A), and TM2 (W611A) NR1 mutations did not affect ethanol sensitivity of heteromeric receptors. In contrast, altering a TM3 phenylalanine to alanine (F639A) reduced the ethanol inhibition of NMDA receptors expressed in oocytes and human embryonic kidney 293 cells. Mutation of the nearby methionine (M641) to alanine did not affect ethanol sensitivity, whereas changing Phe(639) to tryptophan slightly enhanced ethanol inhibition. NR1(F639A) did not alter the agonist potency of glutamate but did produce a leftward shift in the glycine concentration response for receptors containing NR2A and NR2B subunits. NR1(F639A) also reduced the potency of the competitive glycine antagonist 5,7-dichlorokynurenic acid and increased the efficacy of the glycine partial agonist 3-amino-1-hydroxy-2-pyrrolidinone ((+)-HA-966). These results suggest that ethanol may interact with amino acids contained in the TM3 domain of NMDA subunits that are involved in transducing agonist binding to channel opening.

Highlights

N-Methyl-D-aspartate (NMDA)1 receptors are calcium-permeable ion channels expressed by neurons and require both glutamate and glycine for activation
Recent studies from this laboratory have shown that ethanol inhibition of NMDA receptor 1 (NR1)/2A receptors expressed in human embryonic kidney 293 (HEK293) cells is reduced by Fyn tyrosine kinase-mediated phosphorylation of the NR2A subunit as well as by conditions that block calcium-dependent inactivation of NR1/2A receptors [20, 21]
Asterisks indicate residues shown in the NR1 subunit that have been previously assigned as pore-facing by cysteine scanning mutagenesis [29, 30]

Summary

Introduction

N-Methyl-D-aspartate (NMDA) receptors are calcium-permeable ion channels expressed by neurons and require both glutamate and glycine for activation. Ethanol inhibition of NMDAinduced currents in oocytes expressing NR1, NR2A, and NR2C subunits was less than that observed with NR1 and NR2A receptors, suggesting that subunit composition significantly influences overall ethanol sensitivity [19] Recent studies from this laboratory have shown that ethanol inhibition of NR1/2A receptors expressed in human embryonic kidney 293 (HEK293) cells is reduced by Fyn tyrosine kinase-mediated phosphorylation of the NR2A subunit as well as by conditions that block calcium-dependent inactivation of NR1/2A receptors [20, 21]. These data suggest that C-terminal modifications may influence the ethanol sensitivity of the NMDA receptor, it is unlikely that these intracellular domains represent the major site of action for ethanol

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