A Site of Alcohol Action in the Fourth Membrane-associated Domain of the N-Methyl-D-aspartate Receptor

Abstract

The N-methyl-D-aspartate (NMDA) subtype of ionotropic glutamate receptor is an important mediator of the behavioral effects of ethanol in the central nervous system. Although ethanol is known to inhibit NMDA receptors by influencing ion-channel gating, its molecular site of action and the mechanism underlying this effect have not been established. We have previously identified a conserved methionine residue in the fourth membrane-associated domain of the NMDA receptor NR2A subunit (Met 823 ) that influences desensitization and gating of the ion channel. Here we report that this residue plays an important role in mediating the effect of ethanol on the NMDA receptor. Ethanol IC 50 values among functional substitution mutants at this position varied over the range ϳ130 -225 mM. There was a weak correlation between ethanol IC 50 and mean open time of NR2A(Met 823 ) mutants that was dependent on inclusion of the value for the tryptophan mutant. In the absence of this value, there was no trend toward a correlation among the remaining mutants. Desensitization appeared to influence the action of ethanol, because ethanol IC 50 of the mutants was correlated with the steadystate to peak current ratio. With the exception of tryptophan, ethanol sensitivity was significantly related to the molecular volume and hydrophobicity of the substituent. The relation between ethanol sensitivity and the molecular volume and hydrophobicity at this position suggests that this residue interacts with or forms part of a site of ethanol action and that the presence of a tryptophan residue in this site disrupts its ability to interact with ethanol.Ethyl alcohol or ethanol, one of the oldest and most widely abused drugs, produces a well known spectrum of behavioral effects primarily through actions on ion channels in the nervous system. The N-methyl-D-aspartate (NMDA) 1 receptor, a subtype of receptor for the major excitatory neurotransmitter glutamate, is thought to be of particular importance in mediating the effects of alcohols in the mammalian brain. Ethanol inhibits NMDA-activated current in central neurons (1, 2) as well as NMDA-evoked Ca 2ϩ flux, cyclic GMP production, and neurotransmitter release (3-6). Studies using in vivo electrophysiological techniques have also reported ethanol inhibition of NMDA receptors at relevant concentrations (7). Ethanol appears to interact with a novel allosteric site, which is independent of the recognition site for the agonist glutamate or coagonist glycine (6, 8 -12), and reduces the mean open time and opening frequency of the channel (13,14).Presumed alcohol binding sites have been identified on a small number of receptors and ion channels, but the location and nature of these sites vary considerably. Sites of alcohol interaction have been reported to be located in the ion-channel lumen of nicotinic acetylcholine receptors from muscle (15) in a cytoplasmic loop of a Drosophila potassium channel (16), in the cytoplasmic C terminus of G-protein-coupled inwardly rectifying potassium channels (17), and in ...

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

“…The determinants of ethanol sensitivity and receptor function at each of these positions differ markedly from each other. Recently, we found four pairs of positions at the intersubunit interface in the M3 and M4 domains of the GluN1 and GluN2A subunits, including three positions identified previously (Ronald et al, 2001;Ren et al, 2003bRen et al, , 2007, that interact with each other to regulate both ethanol sensitivity and measures of receptor function such as agonist deactivation and desensitization (Ren et al, 2012). In that study, we identified five positions in the GluN1 and GluN2A subunits that had not been previously shown to regulate ethanol sensitivity.…”

“…Reports from our laboratory and others have identified a small number of amino acid residues located in the M3 and M4 domains of NMDA receptor GluN1 and GluN2 subunits that influence ethanol sensitivity and channel function (Ronald et al, 2001;Ren et al, 2003bRen et al, , 2007Honse et al, 2004;Smothers and Woodward, 2006;Xu et al, 2012). The determinants of ethanol sensitivity and receptor function at each of these positions differ markedly from each other.…”

β-N-methylamino-l-alanine enhances neurotoxicity through multiple mechanisms

“…Whole-cell patch-clamp recording was performed as previously described (Ren et al, 2003). Briefly, gigaohm seals were obtained with patch electrodes (series resistance: 2 -10 MΩ, compensation 80%), and cells were voltage-clamped at −50 mV and superfused in extracellular solution containing (in mM): NaCl, 150; KCl, 5; CaCl 2 , 0.2; HEPES, 10; NaHCO 3 , 20; glucose, 10; and sucrose, 10; pH 7.4.…”

Immunosuppressive Effect of Isopropanol: Down-Regulation of Cytokine Production Results from the Alteration of Discrete Transcriptional Pathways in Activated Lymphocytes

“…26). At more physiologically relevant concentrations, alcohols have been shown to induce loss of function of specific proteins, such as ion channels, neurotransmitter receptors, enzymes, and adhesion molecules (27)(28)(29). Structural and biophysical data suggest that binding to the target proteins occurs at discrete sites that are constituted by hydrophobic pockets lined by nonpolar amino acids (26 -29).…”