N-methyl-D-aspartate (NMDA) receptors are glutamate-gated ion channels that play a central role in excitatory neurotransmission, synaptic plasticity, and excitotoxic neuronal injury. Their activation is tightly regulated by co-agonists and modulators, but the structural transitions underlying these processes remain incompletely understood. Here, we employed single-molecule Forster resonance energy transfer (smFRET) to monitor conformational dynamics within the GluN1 (N1) and GluN3 (N3) subunits of NMDA receptors. Specifically, we examined the ligand-binding clefts of N1 and N3, as well as the N3 amino-terminal domain (ATD), under conditions of glycine alone versus glycine plus the glycine-site competitive antagonist CGP. Our results demonstrate distinct ligand-dependent rearrangements. In the presence of glycine alone, the N3 ATD exhibited a more splayed-apart conformation, consistent with the receptor adopting a closed or desensitized state. Conversely, when glycine was co-applied with CGP, the N3 ATD shifted into a more compact arrangement at the top, reflective of the receptor entering an open-channel conformation. These ligand-specific structural transitions were also observed at the N1 and N3 clefts, highlighting coordinated domain movements that shape gating behavior. Together, these findings provide direct evidence that NMDA receptor activation and desensitization involve distinct conformational ensembles that can be differentially stabilized by glycine-site ligands. This work establishes smFRET as a powerful approach to resolve dynamic receptor states and offers new mechanistic insights into the structural basis of NMDA receptor modulation, with implications for therapeutic strategies targeting excitotoxicity in stroke and related neurological disorders.
Verma et al. (2026) studied this question.