Characterization of a Soluble Ligand Binding Domain of the NMDA Receptor Regulatory Subunit NR3A

Abstract

NR3A is expressed widely in the developing CNS of mammals. Coassembly of NR3A with NR1 and NR2 modifies NMDA receptormediated responses, reducing calcium permeability and single-channel conductance. The ligand binding properties of NR3A are unknown but shape the role NR3A plays when incorporated into NMDA receptors. Here, a soluble NR3A ligand binding domain (NR3A S1S2) was constructed based on amino acid sequence alignments with other glutamate receptor ion channels and is expressed in Escherichia coli. After purification by affinity, gel filtration, and ion exchange chromatography, NR3A S1S2 behaves as a monomer even at a concentration of 20 mg/ml, as determined by size-exclusion chromatography and dynamic light scattering. NR3A S1S2 has very high affinity for glycine with an apparent dissociation constant (K d ) of 40 nM, 650-fold less than the K d for NR1. Glutamate, which binds to NR2 subunits, also binds to NR3A, but with very low affinity (K d ϭ 9.6 mM); in contrast, binding of glutamate to NR1 was not detectable even at a 300 mM concentration. The antagonist binding profiles of NR3A and NR1 also show striking differences. 6-Cyano-2,3-dihydroxy-7-nitro-quinoxaline (CNQX), and its analog CGP78608, bind to NR3A S1S2 with low micromolar affinity, whereas for NR1, the affinity of CGP78608 increases 1000-fold compared with CNQX. Other high-affinity NR1 antagonists also show very weak binding to NR3A. Proteolysis protection experiments reveal that CNQX and CGP78608 bind to and stabilize domain 1 of NR3A S1S2 but increase proteolysis of domain 2, indicating that they produce conformational changes distinct from those induced by glycine and D-serine.

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

“…Furthermore, we report here the first functional data displaying selectivity of 5,7-DCKA for GluN1 over GluN3 subunits, which are consistent with previously reported binding studies of the isolated soluble LBDs of GluN1 and GluN3A. 5,7-DCKA has been reported to display 1000-fold selectivity for GluN1 over GluN3A, with binding affinities of 0.54 and 647 M at GluN1 and GluN3A LBDs, respectively (23,30).…”

“…To evaluate the selectivity between the glycine-binding GluN1 and GluN3 subunits, TK40 was also characterized at GluN1(F484A/T518L)/N3A and GluN1(F484A/ T518L)/N3B receptors. 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).…”

“…Thus, glycine site antagonists that discriminate between these glycine sites would be useful tools to selectively study either GluN1 in the NMDA receptor complex or the physiological roles of GluN3-containing NMDA receptors. Studies using the isolated soluble LBDs of GluN1 and GluN3A subunits have revealed a unique selectivity profile for ligand binding to GluN3, which is strikingly different from that of GluN1 (23). For example, glycine site partial agonists display different rank orders of binding affinities at GluN1 and GluN3A.…”

“…5,7-DCKA, L-689,560, and other high affinity glycine site antagonists) exhibit strong selectivity for the isolated GluN1 LBD over the isolated GluN3A LBD, with binding affinities at GluN1 in the nanomolar range versus affinities in the 100 M range for binding to the GluN3A LBD (23). Despite binding of the same endogenous ligand to both GluN1 and GluN3 subunits, glycine has been reported to bind with a 650-fold higher affinity at the isolated GluN3A LBD over the isolated GluN1 LBD, indicating that the glycine-binding site of GluN3 is different from that of GluN1 (23). However, at present, it remains unclear how these binding affinities determined at the soluble LBD translate into potencies at full-length receptors.…”

“…It has previously been observed that binding of the larger agonist D-serine in GluN3A relative to binding of glycine displaces the side chain methyl group of Met 844 laterally and also pushes the side chain carboxyl group of Asp 845 downward. This was speculated to contribute to the 16 and 10 times lower binding affinity of D-serine relative to glycine at the GluN3A-and GluN3B-soluble LBDs, respectively (23,24). Given the larger size of GluN1-selective antagonists like TK40 and 5,7-DCKA, it is possible that these ligands change the position of the interdomain-stabilizing residues differently in the GluN1-and GluN3-containing receptor subtypes and thus indirectly change the dynamics of the ligandreceptor interaction.…”