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 receptor-mediated 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 inEscherichia 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 theK_dfor NR1. Glutamate, which binds to NR2 subunits, also binds to NR3A, but with very low affinity (K_d= 9.6 mm); in contrast, binding of glutamate to NR1 was not detectable even at a 300 mmconcentration. The antagonist binding profiles of NR3A and NR1 also show striking differences. 6-Cyano-2,3-dihydroxy-7-nitro-quinoxaline (CNQX), and its analogCGP78608, bind to NR3A S1S2 with low micromolar affinity, whereas for NR1, the affinity ofCGP78608increases 1000-fold compared with CNQX. Other high-affinity NR1 antagonists also show very weak binding to NR3A. Proteolysis protection experiments reveal that CNQX andCGP78608bind 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 andd-serine.