Subunit-Specific Roles of Glycine-Binding Domains in Activation of NR1/NR3 N-Methyl-d-aspartate Receptors

Abstract

N-Methyl-D-aspartate receptors (NMDARs) composed of NR1 and NR3 subunits differ from other NMDAR subtypes in that they require glycine alone for activation. However, little else is known about the activation mechanism of these receptors. Using NMDAR glycine-site agonists/antagonists in conjunction with functional mutagenesis of the NR1 and NR3 ligand-binding cores, we demonstrate quite surprisingly that agonist binding to NR3 alone is sufficient to activate a significant component of NR1/NR3 receptor currents. Thus, the apo conformation of NR1 in NR1/NR3 receptors is permissive for receptor activation. Agonist-bound NR1 may also contribute to peak NR1/NR3 receptor currents but specifically enables significant NR1/NR3 receptor current decay under the conditions studied here, pre-sumably via a slow component of desensitization. Ligand studies of NR1/NR3 receptors also suggest differential agonist selectivity between NR3 and NR1, as some high-affinity NR1 agonists only minimally activate NR1/NR3 receptors, whereas other NR1 agonists are as potent as glycine. Furthermore, liganded NR3 subunits seem necessary for effective engagement of NR1 in NR1/NR3 receptor activation, suggesting significant interactivity between the two subunits. NR3 subunits thus induce plasticity in NR1 with respect to subunit assembly and ligand binding/channel coupling that is unique among ligand-gated ion channel subunits.

Crystal Structure and Pharmacological Characterization of a Novel N-Methyl-d-aspartate (NMDA) Receptor Antagonist at the GluN1 Glycine Binding Site

“…4, panels B and C and panels E and F). The TK40-mediated potentiation of the glycine-activated current responses and the apparent shift in the bell-shaped concentration-response relationships presumably result from selective inhibition of glycine binding to the GluN1 subunit, which is in agreement with other reports (41,42).…”

“…This bellshaped concentration-response relationship is presumably a result of glycine binding to unblocked GluN3 subunits at low glycine concentrations (rising phase). At higher glycine concentrations, the selective block by TK40 at GluN1 subunits is outcompeted, resulting in diminished receptor current (declining phase), which is in agreement with previous reports (41,42). For glycine-activated responses at wild-type GluN1/N3 receptors, we have previously reported that only negligible current responses (Ͻ10 nA) are observed at GluN1/N3A receptors upon glycine application and that glycine concentrations above 10 M result in diminished receptor current at GluN1/N3B receptors, thereby producing a bell-shaped concentration-response relationship (38).…”

“…Diheteromeric GluN1/N3 receptors are activated by glycine that binds to both the GluN1 and GluN3 subunits (23,24,39,40). Glycine has dual activity at the GluN1/N3 receptors, in that glycine appears to act agonistically at the GluN3 subunit and inhibitory through binding to the GluN1 subunit (41,42), but agonist binding to the GluN3 subunits alone is sufficient to activate the receptor (41, 42). Enhanced glycine activation of GluN1/N3 receptors has initially been shown by disrupting glycine binding to GluN1 via mutation of key amino acids in the GluN1 ligand-binding pocket (41,42).…”

“…Glycine has dual activity at the GluN1/N3 receptors, in that glycine appears to act agonistically at the GluN3 subunit and inhibitory through binding to the GluN1 subunit (41,42), but agonist binding to the GluN3 subunits alone is sufficient to activate the receptor (41, 42). Enhanced glycine activation of GluN1/N3 receptors has initially been shown by disrupting glycine binding to GluN1 via mutation of key amino acids in the GluN1 ligand-binding pocket (41,42). We have recently reported a method to isolate the GluN3 pharmacology of GluN1/N3 receptors by mutating the GluN1 subunit (i.e.…”

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

“…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 endogenous modulator of conventional NMDA receptors is the divalent cation Zn 2ϩ , which allosterically inhibits NR1/NR2 receptors in the submicromolar to micromolar concentration range (15-17). Here, we examined whether Zn 2ϩ also affects the glycine-induced currents of heteromeric NR1/NR3A receptors.…”