Ethanol Inhibition of Constitutively Open N-Methyl-d-Aspartate Receptors

Abstract

N-Methyl-D-aspartate (NMDA) receptors gate a slow and calciumrich component of the postsynaptic glutamate response. Like all ionotropic glutamate receptors, NMDA subunits contain a highly conserved motif (SYTANLAAF) in the transmembrane (TM) 3 domain that is critically involved in channel gating. Mutation of an alanine in this domain (A7; underlined above) results in constitutively open receptors that show reduced sensitivity to several allosteric modulators. In this study, we examined the effects of ethanol, a substance that inhibits NMDA currents via an unknown mechanism, on tonically active NMDA receptors expressed in human embryonic kidney 293 cells. Ethanol (100 mM) inhibited currents from GluN1(A7R)/GluN2A and GluN1(A7R)/GluN2B receptors by approximately 50%, whereas those from GluN1/ GluN2B(A7R) receptors were reduced by less than 10%. In cysteine-substituted GluN1 and GluN2 A7 mutants, estimated ethanol IC 50 values for agonist-gated currents were 101, 117, 103, and 69 mM for GluN1(A7C)/GluN2A, GluN1(A7C)/GluN2B, GluN1/ GluN2A(A7C), and GluN1/GluN2B(A7C) receptors, respectively. After exposure to the thiol-modifying reagent 2-(trimethylammonium)ethyl methanethiosulfonate (MTSET), A7C mutants showed robust agonist-independent currents and reduced sensitivity to ethanol (IC 50 values of 371, 256, 715, and 958 mM, respectively, as above). In contrast, cysteine modification of the ligand-binding domain resulted in constitutively open receptors that showed robust ethanol inhibition. Ethanol inhibition of MTSET-treated GluN1(A7C) receptors was further reduced by TM3/TM4 mutations previously shown to reduce ethanol sensitivity of agonistgated receptors. Overall, these results show that ethanol affects NMDA receptor function at a site distal from agonist binding and appears to exert greater effects via perturbation of GluN2 subunits.

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

“…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.…”

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

“…NMDA receptors are obligate heterotetramers composed of two glycine-binding GluN1 subunits interleaved with two glutamatebinding GluN2 subunits (Traynelis et al, 2010), and results from studies of both native and recombinant NMDA receptors suggest that ethanol acts as an allosteric modulator of receptor function (Chu et al, 1995;Mirshahi and Woodward, 1995;Peoples et al, 1997;Xu et al, 2012). Understanding where and how this allosteric modulation takes place is still not well understood, although recent studies by this laboratory and others have revealed potential sites of ethanol action within the transmembrane (TM) domains of the NMDA receptor (Ronald et al, 2001;Honse et al, 2004).…”

“…Site-directed mutagenesis was performed using the QuikChange XL mutagenesis kit (Agilent Technologies, Santa Clara, CA), and mutants were confirmed by DNA sequencing (GeneWiz, South Plainfield, NJ). NMDA structures were rendered using MacPymol (PyMOL Molecular Graphics System, Version 1.5.0.4; Schrödinger, New York, NY) using a homology model of the GluN1/GluN2A receptor described in a previous study (Xu et al, 2012).…”

“…To determine whether amino acids in nearby TM domains interact with the TM3 F639 residue to influence ethanol inhibition, we used our previously reported GluN1/GluN2A homology model (Xu et al, 2012) to map residues that are in close proximity to F639. This model is based on the crystal structure of the GluA2 homomeric AMPA receptor (Sobolevsky et al, 2009) and has high homology particularly in the TM domains with the recently solved crystal structures of the GluN1/GluN2B receptor (Karakas and Furukawa, 2014;Lee et al, 2014).…”

“…To explore this in a systematic fashion, we used the General Linear Model analysis module in SPSS to analyze how the reduction in ethanol inhibition by the TM3 F639C mutation was affected by cysteine substitutions in the GluN1 TM1 and GluN2 TM4 domains. A single ethanol concentration (100 mM) that is near the IC 50 value for wild-type GluN1/GluN2A receptors (Ren et al, 2003;Xu et al, 2012) was used to screen the various mutants for ethanol inhibition. The results of these experiments are summarized in Fig.…”