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.