Molecular Architecture and Function Mechanism of Tri-heteromeric GluN1-N2-N3A NMDA Receptors

N-methyl-D-aspartate receptors (NMDARs) play a pivotal role in brain development and synaptic function. Previous studies have focused on GluN1-N2 (2A-2D) and GluN1- N3 (3A and 3B) di-heteromeric (di-) NMDARs, leaving the activation mechanism and stoichiometry of GluN1-N2-N3 tri-heteromeric (tri-) NMDARs largely unexplored. In this study, we employed a two-step affinity-tagged chromatography approach to purify recombinantly expressed GluN1-N2A-N3A tri-NMDARs and determined their cryo-EM structure. Based on the proteoliposome single-channel recording, we discovered GluN1-N2A-N3A can be activated upon co-binding of glycine and glutamate, exhibiting two distinct conductance levels. Furthermore, leveraging structural-based click- chemistry, we introduced photo-crosslinker p-azido-phenylalanine (AzF) into the N- terminal domain of GluN2A and GluN2B, enabling the crosslinking with GluN3A subunit both in vitro and in vivo. These findings provide molecular insights into the subunit arrangement, native architecture and activation mechanism of GluN1-N2-N3A tri- NMDARs and also highlight the complexity of NMDAR assembly and function in the brain.