GluN3A and GluN3B are glycine-binding subunits belonging to the NMDA receptor (NMDAR) family that can assemble with the GluN1 subunit to form unconventional receptors activated by glycine alone. Functional characterization of GluN1/GluN3 NMDARs has been difficult. Here, we uncover two modalities that have transformative properties on GluN1/GluN3A receptors. First, we identify a compound, CGP-78608, which greatly enhances GluN1/GluN3A responses, converting small and rapidly desensitizing currents into large and stable responses. Second, we show that an endogenous GluN3A disulfide bond endows GluN1/GluN3A receptors with distinct redox modulation, profoundly affecting agonist sensitivity and gating kinetics. Under reducing conditions, ambient glycine is sufficient to generate tonic receptor activation. Finally, using CGP-78608 on P8-P12 mouse hippocampal slices, we demonstrate that excitatory glycine GluN1/GluN3A NMDARs are functionally expressed in native neurons, at least in the juvenile brain. Our work opens new perspectives on the exploration of excitatory glycine receptors in brain function and development.

NMDA receptors (NMDARs) are glutamate‐gated ion channels that are key mediators of excitatory neurotransmission and synaptic plasticity throughout the central nervous system. They form massive heterotetrameric complexes endowed with unique allosteric capacity provided by eight extracellular clamshell‐like domains arranged as two superimposed layers. Despite an increasing number of full‐length NMDAR structures, how these domains cooperate in an intact receptor to control its activity remains poorly understood. Here, combining single‐molecule and macroscopic electrophysiological recordings, cysteine biochemistry, and in silico analysis, we identify a rolling motion at a yet unexplored interface between the two constitute dimers in the agonist‐binding domain (ABD) layer as a key structural determinant in NMDAR activation and allosteric modulation. This rotation acts as a gating switch that tunes channel opening depending on the conformation of the membrane‐distal N‐terminal domain (NTD) layer. Remarkably, receptors locked in a rolled state display “super‐activity” and resistance to NTD‐mediated allosteric modulators. Our work unveils how NMDAR domains move in a concerted manner to transduce long‐range conformational changes between layers and command receptor channel activity.