Structure and function of GluN1-3A NMDA receptor excitatory glycine receptor channel

Abstract

N-methyl-d-aspartate receptors (NMDARs) and other ionotropic glutamate receptors (iGluRs) mediate most of the excitatory signaling in the mammalian brains in response to the neurotransmitter glutamate. Uniquely, NMDARs composed of GluN1 and GluN3 are activated exclusively by glycine, the neurotransmitter conventionally mediating inhibitory signaling when it binds to pentameric glycine receptors. The GluN1-3 NMDARs are vital for regulating neuronal excitability, circuit function, and specific behaviors, yet our understanding of their functional mechanism at the molecular level has remained limited. Here, we present cryo-electron microscopy structures of GluN1-3A NMDARs bound to an antagonist, CNQX, and an agonist, glycine. The structures show a 1-3-1-3 subunit heterotetrameric arrangement and an unprecedented pattern of GluN3A subunit orientation shift between the glycine-bound and CNQX-bound structures. Site-directed disruption of the unique subunit interface in the glycine-bound structure mitigated desensitization. Our study provides a foundation for understanding the distinct structural dynamics of GluN3 that are linked to the unique function of GluN1-3 NMDARs.

Fig. 1.. Electrophysiological characterization of GluN1a-3A NMDAR constructs.

(A) Chemical structure of the GluN1a-3A NMDAR binding compounds. (B) Whole-cell patch-clamp recordings of HEK293T cells transfected to express WT human GluN1a and WT human GluN3A. Cells were held at −80 mV and exposed to 100 μM glycine for 3 s, with 10 s of rest between each application. (C) Example recording (left) demonstrating the extent of receptor desensitization during a 3-s treatment with glycine. The glycine response was fit with a single exponential (green curve), where the tau value was measured to be 295.4 ± 21.8 ms (mean ± SE). (D) Inhibition of WT GluN1a-3A NMDAR by CNQX. A solution containing 30 μM CNQX was perfused onto the cell for 5 s before exposure to a solution containing both CNQX and glycine. (E) Potentiation of GluN1a-3A NMDAR by CGP-78608. A solution containing 500 nM CGP-78608 was perfused onto the cell for 5 s before treatment with a solution containing both CGP-78608 and glycine. (F) Recording of the GluN1a Phe⁸¹⁰Cys and GluN3A Thr⁶⁷⁵Cys mutants performed as described in (C). (G) Recording of the GluN1a Phe⁸¹⁰Cys ΔCTD and GluN3A Thr⁶⁷⁵Cys ΔCTD mutants used in the cryo-EM studies. All recordings were performed on at least four different cells. A representative recording is shown for each experiment.

Fig. 2.. Cryo-EM structure of CNQX-bound GluN1a-3A NMDAR.

(A) Cryo-EM density (left) and the fitted model. The density was sufficiently resolved for distinguishing GluN1a (magenta) and GluN3A (gray) subunits. GluN1a M4, GluN3A M1′, and the lower lobe of GluN3A ATD (asterisk) are not well resolved. (B) CNQX binding site at the cleft of the GluN1a LBD, showing the density (blue mesh) representing a CNQX molecule (green stick). (C) The GluN1 LBD and GluN3A LBD in the GluN1a-3A NMDAR structure have open cleft conformation similar to the L689,560-bound GluN1 LBD (left, RMSD = 1.15 Å over 267 Cɑ positions, PDB code: 6WHU) and the apo GluN3A LBD (right, RMSD = 0.85 Å over 254 Cɑ positions, PDB code: 4KCD). (D) Subunit arrangement of GluN1a and GluN3A viewed from the extracellular side. Two GluN1a-3A heterodimers (tethered by a black line) are arranged in the 1a-3A-1a′-3A′ pattern in each domain layer. Dimer pairs swap different between the ATD and LBD layers. The TMD channel pore is formed by the M3/M3′ helices.

Fig. 3.. Cryo-EM structure of glycine-bound GluN1a-3A NMDAR.

(A) Cryo-EM density (left) and the fitted model. The density was sufficiently resolved for distinguishing GluN1a (magenta) and GluN3A (gray) subunits. (B) Comparison of the glycine- and CNQX-bound GluN1a-3A NMDARs at LBDs. The upper lobe (D1) residues are superposed, and the rotation angles to align the D2 residues were calculated for GluN1a and GluN3A LBDs. The black rods represent rotational axes. (C) Subunit arrangement of GluN1a and GluN3A viewed from the extracellular side. Two GluN1a-3A heterodimers (tethered by a black line) are arranged in the 1a-3A-1a′-3A′ pattern in the ATD. The GluN1a-3A heterodimer interface is disrupted in LBDs due to ~80° clockwise rotation of the GluN3A subunits compared to the CNQX-bound structure. The TMD channel pore is formed by the M3/M3′ helices.

Fig. 4.. Comparison of GluN1-3A and GluN1-2B NMDARs in different states.

(A) Side views of the receptors in different functional states. COM of ATDs and LBDs are connected by lines. At the bottom of the LBDs are GluN2B Gln⁶⁶² and GluN3A Glu⁷⁷⁶, which are connected to the TMD channel and used to measure the LBD-TMD loop tension. (B and C) Top views of ATDs and LBDs where COMs are connected by lines and angles are measured, showing different subunit orientations. (D and E) Cɑs of GluN2B Gln⁶⁶² and GluN3A Glu⁷⁷⁶ residues (spheres) connected to each other viewed from the top and side.

Fig. 5.. Site-directed mutations at the LBD layer disrupts desensitization.

(A and B) CNQX-bound (A) and glycine-bound (B) GluN1a-3A NMDAR structures viewed from the top of the LBD layer. The rotation transitioning from the CNQX-bound conformation to the glycine-bound conformations results in formation of the new subunit interface around GluN3A His⁷⁸⁷ and Glu⁸¹² with GluN1a Glu⁷⁵¹ and Arg⁷⁵⁵. Specific interactions are shown in the zoom-in panel (right) viewed from the “eye” symbol. (C) Whole-cell patch-clamp recording of WT, GluN3A His⁷⁸⁷Trp, and GluN3A Glu⁸¹²Arg mutants, showing abolishment of desensitization in the mutant channels. (D) Extent of desensitization measured by I_ss/I_max. Error bars represent mean ± SE. Each point represents a single patch.

Fig. 6.. Mechanistic insights into unique features of GluN1-3A NMDAR.

(A) Possible scheme of structural transition from resting (CNQX/CNQX-bound), active (CGP-70608/glycine-bound), and desensitized states (glycine/glycine-bound). While the active-state structure is not available, the open-cleft GluN1 LBD likely maintains the resting-state–like subunit arrangement. Occupation of GluN1 and GluN3A LBDs with glycine results in the ~80° rotation, resulting in desensitization. (B) Dimer of heterodimer interface at the LBD in the CNQX-bound structure (left). The closed-cleft GluN1a LBD was superposed; steric clash occurs between GluN3A loop 1 (cyan) and GluN1a helices F and G (right). There is no interaction between the LBDs and the GluN3A ATD at this site. (C) Equivalent dimer of dimer interface at the LBD of antagonist-bound (L689,560/SDZ220-040, PDB code: 6WHU, left panel) and agonist-bound (glycine/glutamate, PDB code: 6WI1) GluN1-2B NMDARs. The agonist binding does not result in steric clash with GluN2B loop 1 (cyan). Instead, GluN2B loop 1, ATD, and GluN1 LBD interact with each other to form a hub for allosteric coupling via residues such as GluN1a Lys⁵¹⁷ and GluN3A Phe¹⁹⁴ and Leu⁴²⁵.