Supralinear potentiation of NR1/NR3A excitatory glycine receptors by Zn ²⁺ and NR1 antagonist

Abstract

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 M, 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 glycinebinding 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.binding site ͉ ligand-gated ion channel ͉ NMDA receptor ͉ zinc N -methyl-D-aspartate receptors represent a complex family of the tetrameric ionotropic glutamate receptors that have attracted substantial interest because of their unique pharmacology and role in synaptic plasticity and memory formation (1, 2). The diversity of NMDA receptor subtypes derives from a multitude of receptor subunits, which are encoded by seven different genes that have been classified into three subfamilies (NR1, NR2A-D, and NR3A and NR3B) (3). Each subunit shares a common modular design characterized by the extracellular (i) N-terminal domain (NTD), (ii) the S1S2 ligand binding domain (LBD), (iii) an intramembrane region that forms the ion channel, and (iv) an extended intracellular C-terminal tail (4).Conventional NMDA receptors are composed of two glycinebinding NR1 subunits and two glutamate-binding NR2 subunits each (5) and are efficiently activated upon simultaneous binding of both glutamate and glycine (6). Coexpression of the glycinebinding NR3A or NR3B subunit with NR1 and NR2 subunits generates heteromeric receptors composed of NR1, NR2, and NR3 subunits, which also require glutamate and glycine for gating but exhibit altered biophysical properties (7-10). In contrast, recombinant NMDA receptors assembled from two NR1 and two NR3 subunits (11) do not respond to glutamate but are exclusively activated by glycine (''excitatory glycine receptors''; ref. 12). The glycine currents produced by these NR1/NR3 receptors are, however, rather small, although assembly and surface insertion occur with similar efficiency as that of conventional NR1/NR2 receptors (13). Recently, antagonists of the NR1 glycine-binding site were found to markedly enhance the glycine responses of NR1/NR3 receptors (13,14), presumably by preventing rapid receptor desensitization caused by glycine binding to the NR1 subunit.An important endogenou...

Expression of Glycine-Activated Diheteromeric NR1/NR3 Receptors in Human Embryonic Kidney 293 Cells Is NR1 Splice Variant-Dependent

“…Likewise, the splice variant effect was not due to the presence of the N1 cassette because results were similar whether NR1-1a or NR1-1b variants were used. This finding is consistent with results obtained in oocytes reported by Cavara et al (2009) jpet.aspetjournals.org (Awobuluyi et al, 2007;Madry et al, 2007Madry et al, , 2008. In HEK293 cells the NR1 glycine site antagonist, 7-CK, potentiated steady-state currents from NR1-1a and NR1-4a-containing NR1/NR3 receptors to a similar extent.…”

“…A similar action of 7-CK has been reported previously in NR1/NR3A/NR3B receptors (Smothers and Woodward, 2007). This suggests a preferential action of 7-CK to inhibit the NR1 subunit of the NR1/NR3 receptor and is consistent with studies showing that glycine binding to the NR3 subunit results in activation, whereas glycine binding to the NR1 subunit contributes to peak current and promotes desensitization (Awobuluyi et al, 2007;Madry et al, 2007Madry et al, , 2008. Exactly how 7-CK potentiates the currents of NR1/ NR3 receptors is currently unknown.…”

New Insights into the Not-So-New NR3 Subunits of N-Methyl-d-aspartate Receptor: Localization, Structure, and Function

“…In the same study, the reported EC 50 values for glycine activation of NR1/NR3A and NR1/NR3B receptors were approximately 1 and 5 M, respectively. Similar glycine EC 50 (6.5 Ϯ 1.2 M) has been described for NR1/NR3A receptors in oocytes by Madry et al (2007Madry et al ( , 2008. Both studies reported rapid desensitization of glycine-evoked currents at glycine concentrations 3 M and greater.…”

“…Surface triheteromeric NR1/NR2A/ NR3A receptors were detected but not diheteromeric NR1/ NR3A receptors in transiently transfected human embryonic kidney (HEK) 293 cells (Perez-Otano et al, 2001). This is in stark contrast to that reported in X. laevis oocyte expression system Sucher et al, 1995;Wada et al, 2006;Awobuluyi et al, 2007;Madry et al, 2007Madry et al, , 2008. Outside-outside patches pulled from HEK293 cells cotransfected with NR1/NR2A/NR3A cDNAs revealed a low-conductance state (28 pS) in the presence of glutamate and glycine that occurs only when NR3A was cotransfected with other NMDA subunits.…”

A Novel Family of Negative and Positive Allosteric Modulators of NMDA Receptors

“…Constructs were verified by sequencing by the University of Nebraska Medical Center Sequencing Facility. The NTD-deleted NR1 (NR1 ⌬NTD ) and the NTDdeleted NR2 constructs (NR2A ⌬NTD and NR2D ⌬NTD ) were kindly provided by Dr. Bodo Laube (Madry et al, 2008) and Dr. Pierre Paoletti (Rachline et al, 2005), respectively. Plasmids were linearized with NotI (GluN1a, GluN2C, GluN2D, and NR1 ⌬NTD ), EcoRI (GluN2A, GluN2A 2CS1 , and GluN2A 2CS2 ), or SalI (GluN2B, NR2A…”