Abstract
N -methyl-D-aspartate receptors (NMDARs) mediate excitatory signaling essential for synaptic plasticity and memory. Unlike GluN2-containing NMDARs, GluN3-containing receptors are activated solely by glycine, exhibit profound desensitization and paradoxical potentiation by GluN1-selective antagonists, including CGP-78608 (CGP). Although GluN3 NMDARs regulate synapse pruning, excitotoxicity, and are associated with schizophrenia, autism and stroke, their native stoichiometry and gating mechanism poorly defined. Using single-molecule pulldown analysis, we show that native GluN3A receptors are diheteromeric assemblies. Cryo-EM analysis of GluN1/GluN3A receptors in antagonist-bound, pre-active, active, and desensitized states, augmented by electrophysiology and pharmacology experiments, show how glycine activates the receptor solely via GluN3A-dependent conformational changes, opening the gate with ∼2-fold symmetry, and induces a ∼4-fold symmetric desensitized state. CGP-bound GluN1 restricts GluN3A rotation, promoting glycine-induced activation by blocking desensitization. These findings illuminate how CGP potentiates GluN3A receptor activity, place the receptor gating mechanism on a solid structural foundation, and define the molecular basis for pharmacological modulation.