Project Summary The primary objective of this project is to gain a comprehensive understanding of the structures and functions of N-methyl-D-aspartate receptors (NMDARs), which play critical roles in brain functions and development. NMDARs belong to the ionotropic glutamate receptor (iGluR) family, which mediates the majority of excitatory synaptic transmission in mammalian brains. Dysfunctional NMDARs are implicated in neurological disorders and diseases, including Alzheimer’s disease, depression, stroke, schizophrenia, and seizure. They are hetero- tetrameric ligand-gated ion channels comprised of GluN1 and GluN2 and/or GluN3 subunits. The GluN1 and GluN3 subunits bind to glycine, while the GluN2 subunits specifically bind to glutamate. Each NMDAR subunit harbors distinct domains: an amino-terminal domain (ATD), a ligand-binding domain (LBD), a transmembrane domain (TMD), and a carboxyl-terminal domain (CTD). The GluN1-2 NMDARs open their transmembrane ion channels upon binding glycine and glutamate. NMDAR subtypes are defined by different combinations of subunits: four distinct GluN2 subunits (A through D) or two distinct GluN3 subunits (A and B) in conjunction with GluN1. These various subtypes exhibit unique functional properties and display spatiotemporal expression patterns in the brain. In the previous grant cycles, we (1) developed the insect cell expression system, EarlyBac, for effective expression of NMDARs and other complex membrane proteins; (2) elucidated the structure, function, and pharmacology of GluN2C and 2D NMDARs by X-ray crystallography and single-particle cryo-EM; (3) structurally characterized channel blockers and functional antibodies against GluN1-2B NMDARs; and (4) structurally captured pre-active and antagonized states of GluN1-2B NMDARs. Despite these advances, there are still many fundamental questions remaining unanswered, including (a) the lack of a structural basis for the ligand-gated channel activity due to the absence of open NMDAR channel structure and Mg2+-blocked NMDAR structure; (b) the lack of a structural basis for the excitatory glycine receptor, GluN1-3 NMDAR; and (c) the lack of insights into subunit composition of native NMDARs in the brain and structural basis for CTD. We will conduct research aimed at fulfilling these shortfalls. Aim 1 will capture the open gate, and Mg2+ blocked NMDAR structures. Aim 2 will reveal the first structures of GluN1-3A NMDAR in various functional states. Aim 3 will identify NMDAR subtypes in animal brains and capture the CTD, highlighting the NMDAR-calmodulin interaction. These three aims will be achieved by obtaining the structural information of intact NMDAR by single-particle cryo-EM and the CTD fragment by x-ray crystallography. Structure-based mechanistic hypotheses will be examined mainly by electrophysiology. Completing the proposed research aims will represent major advances in understanding the structures and functions of NMDAR subtypes, which will help develop...