Differentiation of Glycine Antagonist Sites ofN-Methyl-d-aspartate Receptor Subtypes

Abstract

The binding site for the co-agonist glycine on N-methyl-D-aspartate (NMDA) receptors has been mapped to the NR1 subunit whereas binding of the principal agonist glutamate is mediated by the NR2 subunits. block, slow activation and inactivation kinetics, and the requirement for glycine as a co-agonist in channel gating (1-6). They play a crucial role in brain development, synaptic plasticity and the pathophysiology of major neurological disorders (7-11). NMDA receptors are assembled from the NR1 subunit in combination with at least one type of NR2 subunit (NR2A-2D) (12-15), giving rise to receptor subtypes with distinct electrophysiological and pharmacological properties (2, 16 -21).Inhibition of NMDA receptors by glycine site antagonists is of major interest for the therapeutic intervention in pathological central nervous system states like stroke, head injury, and epilepsy. At doses evoking remarkable anticonvulsant and neuroprotective effects, glycine site antagonists are less prone to psychotomimetic actions and exhibit less severe motoric and cognitive side effects than glutamate site antagonists (22). Further improvements are expected from the targeting of the glycine site of NMDA receptor subtypes.The NR1 subunit harbors main determinants of the glycine binding site in recombinant receptors as well as in situ. Recombinant homomeric NR1 receptors bind glycine agonists and antagonists (19,23,24), and various amino acid residues involved in high affinity binding have been identified by mutational analysis of the NR1 subunit (25-28). Similarly, in native receptors, the novel glycine antagonist and photoaffinity ligand [ 3 H]CGP 61594 labels exclusively the NR1 subunit of NMDA receptors in brain membranes.2 In contrast, the glutamate binding pocket is formed by the NR2 subunits (29). Interestingly, the properties of the glycine site are influenced by the type of NR2 subunit that is assembled with the NR1 subunit. Glycine displays an up to 10-fold lower potency at NR1/2A receptors than at NR1/2B, NR1/2C, or NR1/2D receptors, and a similar difference in affinity has also been observed for the glycine site agonists D-serine, 1-aminocyclobutanecarboxylic acid, D-alanine, 414 (16,19,21,[30][31][32][33]. However, glycine site antagonists (e.g. L-701,324, L-689,560, L-695,902, CKA) did not clearly differentiate between recombinant NMDA receptor subtypes (23,34). Mutational analysis of the NR1 subunit suggested structural differences in the binding domains of glycine site agonists and antagonists (25)(26)(27)(28). Some of the mutated residues affected the affinity for

Structural Insights Into NMDA Ionotropic Glutamate Receptors via Molecular Modelling

“…Molecular models of both the pore forming region and agonist binding domain of homomeric NMDA-R2C and heteromeric NMDA-R1/R2C channels have been produced. The models provide an explanation for the lack of functional ion channel activity for homomeric NMDA-R2 receptors and provide insights into the differential interactions of these two subunits with glycine and glutamate [18][19][20]. This study extends our earlier work [21], performed before the crystal structures of the agonist binding domain of GluR2 [22] and the KcsA K + channel [23] were available.…”

“…lower than that expected based on percentage amino acid sequence identity [46]. NMDA-R1 subunits are thought to bind glycine whereas the NMDA-R2 subfamily are thought to bind glutamate [18][19][20]. It has been suggested [20] that this difference in binding may arise from the presence of bulky aromatic sidechains in the α-amino binding region of NMDA-R1 subunits.…”

Characterization of the binding of two novel glycine site antagonists to cloned NMDA receptors: evidence for two pharmacological classes of antagonists

“…For example, MDL105,519 does not distinguish between all four NR1‐1a/NR2 forms but in functional studies, L689,560 has a 5 fold selectivity for NR1‐1a/NR2A compared to NR1‐1a/NR2B receptors and notably, the antagonist response curve for NR1‐1a/NR2A deviated from sigmoidal behaviour ( Hess et al ., 1996 ); DKA has an approximate 2 fold lower affinity for NR1‐NR2B, NR1/NR2D compared to NR1/NR2A and NR1/NR2C ( Laurie & Seeburg, 1994 ). More recently, it was shown that [ 3 H]‐CGP 61594, a glycine site antagonist which is also a photoaffinity ligand, preferentially labelled NR2B‐subunit containing NMDA receptors although the incorporated radioactivity was associated with only the M r ∼120 000 NR1 subunit ( Honer et al ., 1998 ). A low affinity reversible binding site for [ 3 H]‐CGP 61594 was found in rat cerebellar membranes.…”

“…A low affinity reversible binding site for [ 3 H]‐CGP 61594 was found in rat cerebellar membranes. It was attributed to binding to NR1/NR2A, NR1/NR2C or both subtypes of receptor since these are the subunits expressed in adult cerebellum ( Honer et al ., 1998 ).…”

Glutamate Receptor Ion Channels: Activators and Inhibitors

“…Potencies for the agonists glycine, D-serine, D-alanine, and 1-amino-carboxycyclobutane are significantly lower at NRlINR2A receptors than receptors composed of NRlINR2B, NR1!NR2C and NRlINR2D (ranked in order of increasing potency; PRIESTLEY et al 1995, LAURIE andSEEBURG 1994;KUTSUWADA et al 1992;BULLER et al 1995;MATSUI et al 1995;HESS et al 1996). Recently a glycine-site antagonist WH]CGP 61594) has been shown to display a high affinity selectively for NR2B-containing receptors (HONER et al 1998). …”