N-Methyl-D-Aspartate (NMDA) receptors are essential for many brain functions. These receptors are heterotetramers typically comprising two GluN1 subunits and two GluN2 subunits. The latter could alternate among four subtypes (N2A-N2D) and determine the functional diversity of NMDA receptors. For example, receptors containing N2C or N2D exhibit 50-fold lower channel open probability (Po) than those containing N2A. Structures of N2A- and N2B-containing receptors have been extensively characterized, providing molecular basis for understanding NMDA receptor function. Here we report the cryo-EM structures of N1-N2D and N1-N2C di-heterotetramers (di-receptors), and N1-N2A-N2C tri-heterotetramer (tri-receptor) at a resolution up to 3.0 angstroms. Structural analysis showed that the bilobate N-terminal domain (NTD) in N2D adopted an intrinsic closed conformation, leading to a compact NTD tetramer in N1-N2D receptor. Functional studies further demonstrated that, in di-receptors containing N2D but not N2A or N2B, crosslinking NTD at the tetrameric interface had no effect on channel activity, while crosslinking ligand-binding domain (LBD) of two N1 protomers significantly elevated Po. Surprisingly, we found that the N1-N2C di-receptors spontaneously oscillated between symmetric and asymmetric conformation. The later one occupied a predominant population, whereby two N2C protomers exhibited distinct conformation. This asymmetry, which was also found to a lesser extent in N1-N2A di-receptor, was further locked by the binding of an N2C-specific allosteric potentiator PYD-106 to a unique binding pocket between NTD and LBD in only one N2C protomer. Finally, N2A and N2C in the N1-N2A-N2C tri-receptor displayed the conformation close to that found in one protomer of N1-N2A and N1-N2C di-receptors, respectively. These findings provide a comprehensive structural understanding of diverse functional properties of major NMDA receptor subtypes.

N-methyl-D-aspartate receptors (NMDARs) are glutamate-gated calcium-permeable excitatory channels. They have attracted great interest as potential targets for the treatment of depression in recent years. NMDARs typically assemble as heterotetramers composed of two GluN1 and two alternative GluN2 (2A-2D) subunits, the latter of which endow various subtypes with diverse gating and pharmacological properties. To date, limited molecules with GluN2 specificity have been identified to show antidepressant effects. Here, we identify a compound termed YY-23 extracted from Rhizoma Anemarrhenae allosterically inhibited the channel activities of GluN2C- or GluN2D-incorporated NMDARs, an effect that was presumably influenced by the S2 segment in the ligand-binding domain of the GluN2 subunit. We found that prefrontal GluN2D-containing NMDARs were predominantly expressed at GABAergic interneurons rather than pyramidal neurons. Furthermore, we revealed that YY-23 suppressed the activity of GluN2D-containing NMDARs and GABAergic neurotransmission in the medial prefrontal cortex (mPFC). As a consequence, this GABAergic disinhibition facilitated the excitatory transmission. Behavioural experiments showed that YY-23 acted as a rapid antidepressant in both stress-naive and stressed animal models, which was abolished in Grin2d-knockout mice. Together, our findings suggest that GluN2D-incorporated NMDARs on GABAergic interneurons might be promising therapeutic targets for the treatment of depression.