Cysteine Substitution of Transmembrane Domain Amino Acids Alters the Ethanol Inhibition of GluN1/GluN2A N-Methyl-d-Aspartate Receptors

Abstract

N-Methyl-D-aspartate receptors (NMDARs) are inhibited by behaviorally relevant concentrations of ethanol, and residues within transmembrane (TM) domains of NMDARs, including TM3 GluN1 phenylalanine 639 (F639), regulate this sensitivity. In the present study, we used cysteine (C) mutagenesis to determine whether there are additional residues within nearby TM domains that regulate ethanol inhibition on NMDARs. GluN1(F639C)/GluN2A receptors were less inhibited by ethanol than wild-type receptors, and inhibition was restored to wild-type levels following treatment with ethanol-like methanethiosulfonate reagents. Molecular modeling identified six residues in the GluN1 TM1 domain (valine V566; serine S569) and the GluN2A TM4 domain (methionine, M817; V820, F821, and leucine, L824) that were in close vicinity to the TM3 F639 residue, and these were individually mutated to cysteine and tested for ethanol inhibition and receptor function. The F639C-induced decrease in ethanol inhibition was blunted by coexpression of GluN1 TM1 mutants V566C and S569C, and statistically significant interactions were observed for ethanol inhibition among V566C, F639C, and GluN2A TM4 mutants V820C and F821C and S569C, F639C, and GluN2A TM4 mutants F821C and L824C. Ethanol inhibition was also reduced when either GluN1 TM1 mutant V566C or S569C was combined with GluN2A V820C, suggesting a novel TM1:TM4 intrasubunit site of action for ethanol. Cysteines substituted at TM3 and TM4 sites previously suggested to interact with ethanol had less dramatic effects on ethanol inhibition. Overall, the results from these studies suggest that interactions among TM1, TM3, and TM4 amino acids in NMDARs are important determinants of ethanol action at these receptors.

Two adjacent phenylalanines in the NMDA receptor GluN2A subunit M3 domain interactively regulate alcohol sensitivity and ion channel gating

“…Both mutant cycle analysis and two-way interaction ANOVA of mean open time values revealed an interaction between the positions, but the molecular mechanism underlying this interaction is unclear. Because GluN2A F636 and F637 are adjacent positions in the M3 domain alpha helix, the nature of this interaction differs from that of interactions we and others have reported between closely apposed side chains on separate M domains at intersubunit interfaces (Ren et al , 2012; Xu et al , 2015). In the latter instance, the distance between the interacting positions, as well as their orientation to one another, would change along with conformational changes associated with ion channel gating.…”

“…Although there is no direct evidence that these positions form binding sites for ethanol, the ability of the positions to interactively and strongly regulate ethanol sensitivity is consistent with this view and provides the best evidence for this to date (Ren et al , 2012; Xu et al , 2015). Stronger evidence for this hypothesis awaits future studies demonstrating persistent alteration of NMDA receptor function by covalently-bound alcohol analogs or localization of ethanol or ethanol analogs to these positions using structural methods such as x-ray crystallography (Sauguet et al , 2013).…”

“…For GluN2A F636 and F637, however, any functional interaction between the positions would be dependent upon additional bonds of one or both positions with nearby residues on other parts of the protein, as discussed above. The presence of these additional bonds appears likely, because positions in the M3 domain have been shown to interact with multiple positions in the M1 and M2 domains in non-NMDA glutamate receptors and NMDA receptors (Ogden and Traynelis, 2013; Lopez et al , 2013; Siegler Retchless et al , 2012; Xu et al , 2015; Wilding et al , 2014). We envision the interactions involving tryptophan as “balancing” this part of the M3 helix, such that the interactions of 636 and 637 with other side chains to alter channel closing rate effectively offset each other (Fig.…”

“…Recent studies have shown that these positions and positions in the GluN1 M4 subunit interact to regulate both ion channel gating and alcohol sensitivity (Ren et al , 2012; Xu et al , 2015). In the present study we report that the side chains at positions 636 and 637 in M3 interact with one another in the regulation of ion channel kinetics and alcohol inhibition.…”

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

“…Our laboratory has reported that two positions in the GluN2A M3 and M4 domains can functionally interact to regulate ethanol sensitivity (Ren et al , 2008), and Smothers et al (2006) identified a pair of positions in the GluN1 subunit that can functionally interact with respect to ethanol inhibition. Positions at the intersubunit M3-M4 domain interfaces interact to regulate ethanol sensitivity and ion channel function (Ren et al , 2012), and can also form three-way interactions with M1 domain residues (Xu et al , 2015). Thus, multiple ethanol-sensitive positions appear to form sites of ethanol action.…”