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N-methyl-d-aspartate (NMDA) receptors composed of glycine-binding GluN1 and GluN3 subunits function as excitatory glycine receptors that respond to agonist application only with a very low efficacy. Binding of glycine to the high-affinity GluN3 subunits triggers channel opening, whereas glycine binding to the low-affinity GluN1 subunits causes an auto-inhibition of the maximal glycine-inducible receptor current (Imax). Hence, competitive antagonists of the GluN1 subunit strongly potentiate glycine responses of wild type (wt) GluN1/GluN3 receptors. Here, we show that co-expression of N-terminal domain (NTD) deleted GluN1 (GluN1(ΔNTD)) and GluN3 (GluN3(ΔNTD)) subunits in Xenopus oocytes generates GluN1/GluN3 receptors with a large increase in the glycine-inducible Imax accompanied by a strongly impaired GluN1 antagonist-mediated potentiation. Affinity purification after metabolic or surface labeling revealed no differences in subunit stoichiometry and surface expression between wt GluN1/GluN3A and mutant GluN1(ΔNTD)/GluN3A(ΔNTD) receptors, indicating a specific effect of NTD deletions on the efficacy of receptor opening. Notably, GluN1/GluN3A(ΔNTD) receptors showed a similar increase in Imax and a greatly reduced GluN1 antagonist-mediated current potentiation as GluN1(ΔNTD)/GluN3A(ΔNTD) receptors, whereas the glycine-induced currents of GluN1(ΔNTD)/GluN3A receptors resembled those of wt GluN1/GluN3A receptors. Furthermore, oxidative crosslinking of the homophilic GluN3A NTD intersubunit interface in mutant GluN1/GluN3A(R319C) receptors caused both a decrease in the glycine-induced Imax concomitantly with a marked increase in GluN1 antagonist-mediated current potentiation, whilst mutations within the intrasubunit region linking the GluN3A NTD to the ligand binding domain had opposite effects. Together these results show that the GluN3A NTD constitutes a crucial regulatory determinant of GluN1/GluN3A receptor function., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Coassembly of the glycine-binding NMDA receptor subunits NR1 and NR3A results in excitatory glycine receptors of low efficacy. Here, we report that micromolar concentrations of the divalent cation Zn(2+) produce a 10-fold potentiation of NR1/NR3A receptor responses, which resembles that seen upon antagonizing glycine binding to the NR1 subunit. Coapplication of both Zn(2+) and NR1 antagonist caused a supralinear potentiation, resulting in a >120-fold increase of glycine-activated currents. At concentrations >50 microM, Zn(2+) alone generated receptor currents with similar efficacy as glycine, implying that NR1/NR3A receptors can be activated by different agonists. Point mutations in the NR1 and NR3A glycine-binding sites revealed that both the potentiating and agonistic effects of Zn(2+) are mediated by the ligand-binding domain of the NR1 subunit. In conclusion, Zn(2+) acts as a potent positive modulator and agonist at the NR1 subunit of NR1/NR3A receptors. Our results suggest that this unconventional member of the NMDA receptor family may in vivo be gated by the combined action of glycine and Zn(2+) or a yet unknown second ligand.

The N-methyl-D-aspartate (NMDA) subtype of ionotropic glutamate receptors (iGluRs) is a tetrameric protein composed of homologous NR1 and NR2 subunits, which require the binding of glycine and glutamate, respectively, for efficient channel gating. The extracellular N-terminal domains (NTDs) of iGluR subunits show sequence homology to the bacterial periplasmic leucine/isoleucine/valine binding protein (LIVBP) and have been implicated in iGluR assembly, trafficking, and function. Here, we investigated how deletion of the NR1- and NR2-NTDs affects the expression and function of NMDA receptors. Both proteolytic cleavage of the NR1-NTD from assembled NR1/NR2 receptors and coexpression of the NTD-deleted NR1 subunit with wild-type or NTD-deleted NR2 subunits resulted in agonist-gated channels that closely resembled wild-type receptors. This indicates that the NTDs of both NMDA receptor subunits are not essential for receptor assembly and function. However, deletion of either the NR1 or the NR2 NTD eliminated high-affinity, allosteric inhibition of agonist-induced currents by Zn2+ and ifenprodil, consistent with the idea that interdomain interactions between these domains are important for allosteric receptor modulation. Furthermore, by replacing the NR2A-NTD with the NR2B NTD, and vice versa, the different glycine affinities of NR1/NR2A and NR1/NR2B receptors were found to be determined by their respective NR2-NTDs. Together, these data show that the NTDs of both the NR1 and NR2 subunits determine allosteric inhibition and glycine potency but are not required for NMDA receptor assembly.

Calcium-permeable N-methyl-d-aspartate (NMDA) receptors are tetrameric cation channels composed of glycine-binding NR1 and glutamate-binding NR2 subunits, which require binding of both glutamate and glycine for efficient channel gating. In contrast, receptors assembled from NR1 and NR3 subunits function as calcium-impermeable excitatory glycine receptors that respond to agonist application only with low efficacy. Here, we show that antagonists of and substitutions within the glycine-binding site of NR1 potentiate NR1/NR3 receptor function up to 25-fold, but inhibition or mutation of the NR3 glycine binding site reduces or abolishes receptor activation. Thus, glycine bound to the NR1 subunit causes auto-inhibition of NR1/NR3 receptors whereas glycine binding to the NR3 subunits is required for opening of the ion channel. Our results establish differential roles of the high-affinity NR3 and low-affinity NR1 glycine-binding sites in excitatory glycine receptor function.