Although the N-methyl-D-aspartate (NMDA) receptor plays a critical role in the central nervous system, many questions remain regarding the relationship between its structure and functional properties. In particular, the involvement of ligandbinding domain closure in determining agonist efficacy, which has been reported in other glutamate receptor subtypes, remains unresolved. To address this question, we designed dual cysteine point mutations spanning the NR1 and NR2 ligandbinding clefts, aiming to stabilize these domains in closed cleft conformations. Two mutants, E522C/I691C in NR1 (EI) and K487C/N687C in NR2 (KN) were found to exhibit significant glycine-and glutamate-independent activation, respectively, and co-expression of the two subunits produced a constitutively active channel. However, both individual mutants could be activated above constitutive levels in a concentration-dependent manner, indicating that cleft closure does not completely prevent agonist association. Interestingly, whereas the NR2 KN disulfide was found to potentiate channel gating and M3 accessibility, NR1 EI exhibited the opposite phenotype, suggesting that the EI disulfide may trap the NR1 ligand-binding domain in a lower efficacy conformation. Furthermore, both mutants affected agonist sensitivity at the opposing subunit, suggesting that closed cleft stabilization may contribute to coupling between the subunits. These results support a correlation between cleft stability and receptor activation, providing compelling evidence for the Venus flytrap mechanism of glutamate receptor domain closure. Ionotropic glutamate receptors (iGluRs)2 are key mediators of excitatory neurotransmission, contributing to both neuronal development and adult neuroplasticity (1, 2). In particular, the N-methyl-D-aspartate (NMDA) receptor subclass is responsible for initiating activity-dependent changes in synaptic strength, proposed to form the molecular basis of learning and memory (3). Additionally, NMDA receptors have been implicated in the pathogenesis of numerous neurological disorders, including schizophrenia and Alzheimer disease, as well as neurodegeneration following ischemia or brain injury (4 -6). Despite their enormous therapeutic potential, clinical use of NMDA receptor antagonists has been limited by significant neurological side effects. However, D-cycloserine (DCS), a well tolerated partial agonist, has shown therapeutic promise as a cognitive enhancer in Alzheimer disease and head trauma recovery (7,8). Therefore, the mechanism by which glutamate receptors distinguish partial agonists from full agonists is of both scientific and medical interest.The iGluR family is composed of three subclasses, ␣-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA), kainate, and NMDA receptors, all of which share the same modular domain structure. Two discontinuous segments, S1 and S2, form the ligand binding domain (LBD), whereas the ion channel portion contains three putative transmembrane segments, M1, M3, and M4, and a re-entrant loop, M2. NMDA r...

N-Methyl-D-aspartate (NMDA) receptors play a critical role in both development of the central nervous system and adult neuroplasticity. However, although the NMDA receptor presents a valuable therapeutic target, the relationship between its structure and functional properties has yet to be fully elucidated. To further explore the mechanism of receptor activation, we characterized two gain-of-function mutations within the NR1 M3 segment, a transmembrane domain proposed to couple ligand binding and channel opening. Both mutants (A7Q and A7Y) displayed significant glycine-independent currents, indicating that their M3 domains may preferentially adopt a more activated conformation. Substituted cysteine modification experiments revealed that the glycine binding clefts of both A7Q and A7Y are inaccessible to modifying reagents and resistant to competitive antagonism. These data suggest that perturbation of M3 can stabilize the ligand binding domain in a closed cleft conformation, even in the absence of agonist. Both mutants also displayed significant glutamate-independent current and insensitivity to glutamate-site antagonism, indicating partial activation by either glycine or glutamate alone. Furthermore, A7Q and A7Y increased accessibility of the NR2 M3 domain, providing evidence for intersubunit coupling at the transmembrane level and suggesting that these NR1 mutations dominate the properties of the intact heteromeric receptor. The equivalent mutations in NR2 did not exhibit comparable phenotypes, indicating that the NR1 and NR2 M3 domains may play different functional roles. In summary, our data demonstrate that the NR1 M3 segment is functionally coupled to key structural domains in both the NR1 and NR2 subunits.

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