Structural insights into NMDA receptor pharmacology

Abstract

N-methyl-d-aspartate receptors (NMDARs) comprise a subfamily of ionotropic glutamate receptors that form heterotetrameric ligand-gated ion channels and play fundamental roles in neuronal processes such as synaptic signaling and plasticity. Given their critical roles in brain function and their therapeutic importance, enormous research efforts have been devoted to elucidating the structure and function of these receptors and developing novel therapeutics. Recent studies have resolved the structures of NMDARs in multiple functional states, and have revealed the detailed gating mechanism, which was found to be distinct from that of other ionotropic glutamate receptors. This review provides a brief overview of the recent progress in understanding the structures of NMDARs and the mechanisms underlying their function, focusing on subtype-specific, ligand-induced conformational dynamics.

Keywords: glutamate receptor; neuropharmacology; structure.

Figure 1.. N-methyl-d-aspartate receptor (NMDAR) architecture and assembly.

(A) Linear representation of the amino-terminal domain (ATD), ligand-binding domain (LBD), transmembrane domain (TMD), and C-terminal domain (CTD) within an NMDAR subunit polypeptide chain. The LBD comprises two separate polypeptides (S1 and S2) that fold into a clamshell-like structure. The TMD is formed by three transmembrane helices (M1, M3, and M4) and a membrane-reentrant loop (M2). (B) The chemical structures of the NMDAR ligands Ifenprodil (allosteric inhibitor), SDZ220-040 (competitive antagonist), and ketamine (open channel blocker). (C) Top: Common domain organization of NMDARs. The ATD and LBD are formed from two regions named R1 and R2 (ATD) and D1 and D2 (LBD). Agonists and antagonists docking between the LBD D1–D2 lobes are indicated (‘ligand’, yellow). The LBD and TMD are connected via three linkers, namely, S1–M1 (D2–M1), M3–S2 (D2–M3), and S2–M4 (D1–M4) (‘linkers’). Bottom: NMDAR subunit combinations. NMDARs are assembled as di-heteromeric or tri-heteromeric complexes. The latter contain two different GluN2 subunits (e.g. GluN1/GluN2A/GluN1/GluN2B). (D) Cryo-electron microscopy (Cryo-EM)-resolved structure of an intact di-heterotetrameric GluN1/GluN2B NMDAR in complex with S-(+)-ketamine, with the CTD deleted (PDB ID: 7SAC), with overviewing different NMDAR ligand-binding sites. The GluN1 and GluN2 subunits are in gray and blue, respectively. The LBD–TMD linkers are highlighted (gray box). (E) Ligand-binding sites. Top: Ifenprodil binding to the GluN1–GluN2B ATD heterodimer interface (PDB ID: 5EWJ); middle: glycine and SDZ220-040 binding to the ligand-binding sites on GluN1 and GluN2B, respectively (PDB ID: 6USV); bottom: ketamine binding in the ion channel pore formed by the M3 helices of GluN1 and GluN2B (PDB ID: 7SAC).

Figure 2.. Structural conservation of surface-exposed amino acids in NMDARs.

(A) Linear representation of the GluN1 polypeptide chain showing eight splice variants. The different GluN1 splice variants arise from the alternative splicing of exons 5, 21, and 22/22′, yielding the N1, C1, and C2/C2′ cassettes. (B) The conservation pattern of surface-exposed amino acids in human GluN2A–D subunits based on the structures of GluN1/GluN2A (PDB ID: 7EU7), GluN1/GluN2B (7EU8), GluN1/GluN2C (8E93), and GluN1/GluN2D (8E96). The color-coding bar shows the coloring scheme generated using Al2CO. Conserved amino acids are in purple, residues of average conservation are in white, and variable amino acids are in turquoise. The following NMDAR amino acid sequences were used: GluN1 (Uniprot # Q05586), GluN2A (Q12879), GluN2B (Q13224), GluN2C (Q14957), GluN2D (Q14957), GluN3A (Q8TCU5), and GluN3B (O60391).

Figure 3.. NMDAR activation and inhibition.

(A) Schematic representation of NMDAR activation. Agonist binding at both GluN1 and GluN2 ligand-binding domains (LBDs) triggers clamshell closure. Clamshell closure generates tension in the LBD-M3 linkers and pulls the M3 helices, thus opening the pore. (B) Magnified views of the superposition of helix E in the LBD D2 lobe, pore-lining M3, and LBD-M3 linkers in GluN1 and GluN2B subunits in the agonist-bound ‘non-active 1’ (PDB ID: 6WHS) and the ‘active’ (PDB ID: 6WI1) structures. The spheres indicate the residue GluN2 (Q662) with cross-dimer distances. (C) Left: The structure of the GluN1/GluN2B NMDAR in the ‘active’ state highlighting the domain or subunit interfaces: ATD^GluN1/ATD^GluN2, ATD^GluN2–LBD^GluN2, ATD^GluN2–LBD^GluN1, and LBD^GluN1–LBD^GluN2 (cyan, pink, green, and orange, respectively). GluN1–GluN2B ATD heterodimer cysteine cross-linking (GluN1^A175C/2B^Q180C) is shown. Right: The application of a bifunctional cross-linker [1,4-butanediyl bismethanethiosulfonate (M4M)] to the GluN1^A175C/2B^Q180C NMDAR mutant in the presence of glycine and glutamate potentiates the microscopic current as measured by two-electrode voltage clamp recording. Modified from Tajima et al. [31]. (D) Magnified view of the ATD–LBD and LBD–LBD interfaces in the GluN1/GluN2B NMDAR structure. (E) Extracellular view of the ion channel in an active GluN1/GluN2B NMDAR (PDB ID: 6WI1). (F) The channel blocker binding site and residues involved in the interaction. Modified from Chou et al. [35].

Figure 4.. Schematic summary of NMDAR modulation by ligands and Ions.

Antagonism: The binding of competitive antagonists causes the ligand-binding domain (LBD) bi-lobe to close, resulting in the closing of the ion channel pore. Inhibition: Some allosteric inhibitors such as ifenprodil and Ro25-6981 bind at the GluN1–GluN2 amino-terminal domain (ATD) heterodimer interface and close the ATD bi-lobe, blocking the receptors in inactive conformations. Blocking: Channel blockers inhibit NMDAR by occluding the ion channel. Inhibition (Zn²⁺/H⁺-involvement): Zn²⁺ and protons induce clamshell closure in the GluN2 ATD and close the ion channel. Active/Open: Glycine/glutamate binding triggers conformational changes in the LBDs of GluN1/GluN2 NMDARs. The conformational rearrangement of the GluN1–GluN2 ATD couples the GluN1–GluN2 LBD with downstream gating.

Figure 5.. Chemical structures of antagonists, open channel blockers, and allosteric modulators of NMDARs.

The chemical structures and the subtype specificity are shown.