Research Article

Cryo-EM structures of the triheteromeric NMDA receptor and its allosteric modulation

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Science  24 Mar 2017:
Vol. 355, Issue 6331, eaal3729
DOI: 10.1126/science.aal3729

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Added complexity in an asymmetric receptor

N-methyl-d-aspartate receptors (NMDARs) are heterotetrameric ion channels that initiate chemical and electrical signals in postsynaptic cells. They play key roles in brain development and function and are the targets of drugs for treating neurological disorders such as schizophrenia, depression, and epilepsy. For the channel to open, it must bind glutamate and glycine and release a blocking magnesium ion. Most NMDARs have three different subunits that bind glycine and glutamine, but structural studies have focused on tetramers of only two subunits. Lü et al. determined the structure of triheteromeric NMDAR. The structural studies show how having three different subunits modifies receptor symmetry and subunit interactions and increases the complexity of receptor regulation.

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Structured Abstract


Chemical neurotransmission is fundamental to communication between neurons and to the alternation of the “strength” of neuron-to-neuron connections in an experience-dependent manner. N-methyl-d-aspartate receptors (NMDARs) are neurotransmitter-activated ion channels that act as Hebbian-like coincidence detectors, requiring the binding of glutamate and glycine together with the voltage-dependent relief of magnesium block from the ion channel pore. Because the open NMDAR ion channel pore conducts both monovalent ions and Ca2+, not only does the activation of NMDARs elicit an electrical signal but also the entry of Ca2+ provides a chemical signal, initiating intracellular calcium-dependent signaling processes. NMDARs are ubiquitously dispersed throughout the central nervous system, play crucial roles in brain development and function, and are the targets of clinically relevant drugs for treatment of mild cognitive impairment, schizophrenia, depression, and epilepsy.

Diversity in NMDAR function is the consequence of receptor assembly as heterotetramers with different receptor subunit combinations found in distinct brain regions. The palette of NMDAR building blocks includes the extensively studied glycine-binding and glutamate-binding GluN1 and GluN2A to -D subunits, respectively, together with the rather enigmatic glycine-binding GluN3A and -B subunits. The canonical NMDAR is composed of two GluN1 subunits and two GluN2 subunits, where the two GluN2 subunits can be either identical or different, thus giving rise to diheteromeric or triheteromeric NMDARs, respectively. Despite the prevalence of triheteromeric receptors throughout the brain, such as the GluN1/GluN2A/GluN2B receptor, the dominant NMDAR in the hippocampus and cortex, physiological and structural studies on NMDARs have been almost exclusively restricted to diheteromeric receptors. However, triheteromeric NMDARs are endowed with channel gating kinetics and receptor pharmacology distinct from the GluN2A- and GluN2B-containing diheteromeric receptors. Furthermore, the triheteromeric receptor is uniquely modulated by GluN2A- and GluN2B-specific allosteric antagonists.


To determine how incorporation of two different GluN2 subunits alters receptor symmetry and subunit-subunit interactions, we resolved the structure of the GluN1/GluN2A/GluN2B receptor by single-particle cryogenic electron microscopy. Because the GluN2A and GluN2B subunits are structurally related, we used a GluN2B-specific Fab to unambiguously distinguish the two GluN2 subunits. To understand the molecular basis for the action of GluN2B-specific allosteric modulator in the context of a GluN2A subunit, we carried out structural studies in the presence or absence of the GluN2B-specific allosteric antagonist Ro 25-6981 (Ro).


The triheteromeric NMDAR adopts a bouquet-like shape assembled as a GluN1/GluN2A/GluN1/GluN2B heterotetramer, with each subunit at the canonical A/B/C/D positions, respectively. The amino-terminal domains (ATDs) and ligand-binding domains (LBDs) define a large, synaptically localized extracellular structure, and the transmembrane domains (TMDs) form the ion-conducting channel. Throughout the extracellular regions, the receptor displays a “dimer-of-dimers” arrangement, with a swapping of domains between the ATD and LBD layers.

The presence of GluN2A and GluN2B subunits in the triheteromeric receptor disrupts the 2-fold symmetry in the ATD and LBD layers and the pseudo–4-fold symmetry in the TMD layer. Within the ATD layer, the GluN2A and GluN2B ATDs adopt “closed” and “open” clefts, respectively. Upon binding Ro, the GluN2B ATD clamshell transitions from an open to a closed conformation. Compared with the GluN2B subunit, the GluN2A ATD interacts more extensively with the GluN1 subunit within the ATD heterodimer and thus is poised to modify the conformational properties of its GluN1 ATD partner to a greater extent than the GluN2B. At the ATD-LBD interface, the GluN2A ATD caps the LBD layer, participating in extensive interactions with the LBD layer. By contrast, the GluN2B ATD is located farther away from the LBD layer. In the LBD layer, the GluN2A LBD interacts extensively with both GluN1 subunits, whereas the GluN2B LBD is primarily coupled to the GluN1 subunit within the LBD heterodimer. Therefore, the GluN2A subunit interacts more extensively with GluN1 subunits throughout the receptor, in comparison with the GluN2B subunit, consistent with the predominant role of the GluN2A subunit in sculpting the ion channel kinetics of the triheteromeric receptor.


The structural studies reveal the architecture of the triheteromeric receptor, define the molecular action of GluN2B-specific modulator Ro, and show how the GluN2A and GluN2B subunits participate in distinct interactions throughout the receptor assembly.

Schematic representation of the triheteromeric NMDAR.

(A) NMDARs are localized in the postsynapse. (B) Binding of glycine to the GluN1 subunits and glutamate to the GluN2 subunits promotes closure of the LBD “clamshells” and opening of the ion channel. (C) Allosteric antagonists zinc and Ro bind to the GluN2A and GluN2B subunits, respectively, promoting channel closure by altering the conformation of the LBD layer. Arrows show ATD cleft closure and possible LBD movement.


N-methyl-d-aspartate receptors (NMDARs) are heterotetrameric ion channels assembled as diheteromeric or triheteromeric complexes. Here, we report structures of the triheteromeric GluN1/GluN2A/GluN2B receptor in the absence or presence of the GluN2B-specific allosteric modulator Ro 25-6981 (Ro), determined by cryogenic electron microscopy (cryo-EM). In the absence of Ro, the GluN2A and GluN2B amino-terminal domains (ATDs) adopt “closed” and “open” clefts, respectively. Upon binding Ro, the GluN2B ATD clamshell transitions from an open to a closed conformation. Consistent with a predominance of the GluN2A subunit in ion channel gating, the GluN2A subunit interacts more extensively with GluN1 subunits throughout the receptor, in comparison with the GluN2B subunit. Differences in the conformation of the pseudo-2-fold–related GluN1 subunits further reflect receptor asymmetry. The triheteromeric NMDAR structures provide the first view of the most common NMDA receptor assembly and show how incorporation of two different GluN2 subunits modifies receptor symmetry and subunit interactions, allowing each subunit to uniquely influence receptor structure and function, thus increasing receptor complexity.

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