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

Abstract

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.

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

“…Previous studies showed that ethanol inhibits NMDA receptors by altering ion channel gating, primarily by decreasing mean open time of the channel, and studies from our laboratory reported that mutations at ethanol-sensitive positions in the GluN2A subunit also strongly influence ion channel gating kinetics (Ren et al , 2003a; Ren et al , 2007; Ren et al , 2008; Honse et al , 2004; Ren et al , 2013). In this study, we first compared glutamate peak and steady-state EC 50 values between GluN2B wild-type and GluN2B(F637W) mutant subunits.…”

“…For a series of mutants at GluN2A(F636), glutamate EC 50 values for steady-state current were highly correlated with those for peak current, but not with values of I ss :I p (Ren et al , 2013). In the present study, we observed that peak current glutamate EC 50 values were strongly correlated with steady-state current glutamate EC 50 values ( R 2 = 0.95, P < 0.0001; Fig 7A).…”

“…Previous studies from this laboratory have identified and characterized a number of ethanol-sensitive positions in these domains of the GluN2A subunit (Fig. 1B) (Honse et al , 2004; Ren et al , 2003b, 2007, 2013). In this study, we initially constructed tryptophan and alanine substitutions at positions that correspond to ethanol-sensitive residues in the GluN2A subunit: Phe637, Phe638, Met824, and Gly826.…”

“…Linear relations of mean values of log IC 50 , log EC 50 , or maximal steady-state to peak current ratio (I SS : I P ) for the various mutants in GluN2A and GluN2B subunit were made using linear regression analysis. Values of log IC 50 , log EC 50 , and maximal steady-state to peak current ratio (I SS : I P ) for GluN2A mutants are from previous studies (Ren et al , 2003a; Honse et al , 2004; Ren et al , 2007; Ren et al , 2013). Time constants (τ) of deactivation were determined from fits of the current decay following the removal of glutamate (in the continued presence of glycine) to an exponential function using Clampfit (Axon Instruments).…”

“…The NMDA receptor is a heterotetramer formed from two obligatory GluN1 subunits and two GluN2 subunits, of which four different isoforms are expressed in the brain (GluN2A-GluN2D; (Dingledine et al , 1999)). Previous studies in our and other laboratories have identified and characterized a number of ethanol-sensitive positions in the M3 and M4 domain of the GluN1 and GluN2A subunit (Honse et al , 2004; Ren et al , 2003a, 2003b, 2007, 2012, 2013; Ronald et al , 2001; Smothers and Woodward, 2006; Xu et al , 2012). Although the GluN2A subunit-containing NMDA receptor predominates in the mammalian brain, the GluN2B subunit has comparable ethanol sensitivity (Allgaier, 2002; Kuner et al , 1993; Masood et al , 1994; Popp et al , 1998; Smothers et al , 2001) and plays an important role in alcohol action (Boyce-Rustay and Holmes, 2005; Izumi et al , 2005; Kash et al , 2008, 2009; Nagy, 2004; Wang et al , 2007; Wills and Winder, 2013).…”