Influence of a threonine residue in the S2 ligand binding domain in determining agonist potency and deactivation rate of recombinant NR1a/NR2D NMDA receptors

Abstract

NR1/NR2D NMDA receptors display unusually slow deactivation kinetics which may be critical fortheirroleasextrasynapticreceptors.Athreoninetoalaninepointmutationhasbeeninserted at amino acid position 692 of the NR2D subunit (T692A). Recombinant NR1a/NR2D(T692A) NMDA receptors have been expressed in Xenopus laevis oocytes and their pharmacological and single-channel properties examined using two-electrode voltage-clamp and patch-clamp recording techniques. Glutamate dose-response curves from NR1a/NR2D(T692A) receptor channels produced an approximately 1600-fold reduction in glutamate potency compared to wild-type NR1a/NR2D receptors. There was no change in Hill slopes or gross reduction in mean maximal currents recorded in oocytes expressing either wild-type or mutant receptors. The mutation did not affect the potency of the co-agonist glycine. The shifts in potency produced by NR2D(T692A) containing receptors when activated by other glutamate-site agonists such as aspartate or NMDA were 30-to 60-fold compared to wild-type. Single-channel conductance levels of NR1a/NR2D(T692A) mutant receptors were indistinguishable from wild-type NR2D-containing channels. Additionally NR1a/NR2D(T692A) receptors showed the transitional asymmetry that is characteristic of NR2D-containing NMDA receptors. Rapid applications of glutamate on outside-out patches containing NR1a/NR2D(T692A) receptors produced macroscopic current deactivations that were about 60-fold faster than wild-type NR1a/NR2D receptors. Our results suggest that this conserved threonine residue plays a crucial role in ligand binding to NMDA NR2 receptor subunits and supports the idea that the slow decay kinetics associated with NR1a/NR2D NMDA receptors can be explained by the slow dissociation of glutamate from this NMDA receptor subtype.

Structural Features of the Glutamate Binding Site in Recombinant NR1/NR2A N-Methyl-D-aspartate Receptors Determined by Site-Directed Mutagenesis and Molecular Modeling

“…Previous studies have characterized the properties of glutamate-activated NR1/NR2A(T671A) receptors and the homologous mutation in NR2D-containing NMDA receptors at both the whole-cell and single-channel level (Anson et al, 1998(Anson et al, , 2000Chen et al, 2004). In the present study, we find that the potencies for all four agonists were decreased by this mutation, with effects on glutamate potency being the largest (848-fold; Table 1).…”

“…For the GluR2 and NR2A subunits, the numbering refers to the mature protein, whereas those numbers given for NR1 include the signal peptide (Armstrong et al, 1998;Armstrong and Gouaux, 2000;Furukawa and Gouaux, 2003). A number of studies have reported that mutations of these residues in various NR2 subunits can alter glutamate potency (Williams et al, 1996;Laube et al, 1997;Anson et al, 1998;Chen et al, 2004;Kalbaugh et al, 2004; for review, see Erreger et al, 2004).…”

Equilibrium Constants for (R)-[(S)-1-(4-Bromo-phenyl)-ethylamino]-(2,3-dioxo-1,2,3,4-tetrahydroquinoxalin-5-yl)-methyl]-phosphonic Acid (NVP-AAM077) Acting at Recombinant NR1/NR2A and NR1/NR2B N-Methyl-d-aspartate Receptors: Implications for Studies of Synaptic Transmission

“…Notwithstanding, this study is the first to report equilibrium constants for NVP-AAM077 action at NR1/NR2A and NR1/NR2B NMDA receptors and to demonstrate that its action is indeed competitive in nature and is surmountable by increasing the concentration of glutamate used to activate receptors. In addition, our results show that at least one of the residues (Thr671) that interacts directly with glutamate in the ligand binding domain (for review, see Chen and Wyllie, 2006; also see Anson et al, 1998Anson et al, , 2000Chen et al, 2004Chen et al, , 2005Furukawa et al, 2005) also influences NVP-AAM077 binding. The NR2A(T671A) mutation has previously been shown to reduce D-AP5 potency (Anson et al, 1998), suggesting that this residue plays an important role in competitive antagonist binding as well as agonist binding in NR2A NMDA receptor subunits.…”

“…The ligand binding domain of such subunits is formed by amino acids located in the S1 region (located between the amino terminal domain and the first membraneassociated region) and the S2 region (located between the third and fourth membrane-associated regions). In all glutamate binding subunits, a conserved serine-threonine motif is found in the S2 domain, and both of these amino acids are known, from crystallographic studies (Furukawa et al, 2005) and supported by data from functional studies (for review, see Chen and Wyllie, 2006; also see Anson et al, 1998;Chen et al, 2004Chen et al, , 2005, to be directly involved in hydrogen bonding with glutamate when it occupies the binding pocket. The partial sequence (and alignment) of residues found in the S2 ligand binding domain is shown below the linear representation of the receptor subunit.…”

The Integrity of the Glycine Co-agonist Binding Site of N-Methyl-d-aspartate Receptors Is a Functional Quality Control Checkpoint for Cell Surface Delivery

“…It is thought that these two lobes form a Venus flytrap or clamshell structure that is opened following the binding of either glycine (NR1) or glutamate (NR2). Functional studies of wild-type and point-mutated subunits expressed in Xenopus oocytes (3)(4)(5)(6)(7)(8)(9)(10)(11) or mammalian cells (12) have identified within these domains key amino acids that are important mediators for the high affinity binding of glycine (2)(3)(4)(5)(6) and glutamate (7)(8)(9)(10)(11)(12). More recently, the crystal structures of the soluble NMDA receptor NR1 S1-S2 and NR2 S1-S2 loop constructs in the presence of the appropriate agonist were resolved, consolidating the findings from the mutagenesis experiments (13,14).…”