Structure, function, and allosteric modulation of NMDA receptors

Abstract

NMDA-type glutamate receptors are ligand-gated ion channels that mediate a Ca2+-permeable component of excitatory neurotransmission in the central nervous system (CNS). They are expressed throughout the CNS and play key physiological roles in synaptic function, such as synaptic plasticity, learning, and memory. NMDA receptors are also implicated in the pathophysiology of several CNS disorders and more recently have been identified as a locus for disease-associated genomic variation. NMDA receptors exist as a diverse array of subtypes formed by variation in assembly of seven subunits (GluN1, GluN2A-D, and GluN3A-B) into tetrameric receptor complexes. These NMDA receptor subtypes show unique structural features that account for their distinct functional and pharmacological properties allowing precise tuning of their physiological roles. Here, we review the relationship between NMDA receptor structure and function with an emphasis on emerging atomic resolution structures, which begin to explain unique features of this receptor.

© 2018 Hansen et al.

Figures

Figure 1.

Functional classes of iGluRs. (A) iGluRs are divided into AMPA, kainate, and NMDA receptors with multiple subunits cloned in each of these functional classes. (B) EPSCs from central synapses can be divided into fast AMPA or slow NMDA receptor–mediated components in the absence of Mg2+ using the AMPA receptor antagonist CNQX or the NMDA receptor antagonist AP5. The figure is adapted from Traynelis et al. (2010). (C) The relationships between NMDA receptor current response and membrane potential (i.e., holding potential) in the presence and absence of 100 µM extracellular Mg2+ reveal the voltage-dependent Mg2+ block, which is relieved as the membrane potential approaches 0 mV (i.e., with depolarization). Data are from Yi et al. (2018).

Figure 2.

Subunit stoichiometry and subunit arrangement of GluN1/2 NMDA receptors. The crystal structure of the intact GluN1/2B NMDA receptor (the intracellular CTD omitted from structure; Protein Data Bank accession no. 4PE5; Karakas and Furukawa, 2014) definitively demonstrated that GluN1 and GluN2 subunits assemble as heterotetramers with an alternating pattern (i.e., 1-2-1-2). The NMDA receptor is therefore comprised of two glycine-binding GluN1 and two glutamate-binding GluN2 subunits (i.e., GluN1/2 receptors) that form a central cation-permeable channel pore.

Figure 3.

Expression and functional properties of NMDA receptor subtypes determined by the GluN2 subunit. (A) Autoradiograms obtained by in situ hybridizations of oligonucleotide probes to parasagittal sections of rat brain at indicated postnatal (P) days reveal distinct regional and developmental expression of GluN2 subunits. Fig. 3 A is modified from Akazawa et al., 1994 with permission from the Journal of Comparative Neurology. (B) Whole-cell patch-clamp recordings of responses from recombinant diheteromeric NMDA receptor subtypes expressed in HEK293 cells. The receptors are activated by a brief application of glutamate (1 ms of 1 mM glutamate) indicated by the open tip current in the upper trace. Fig. 3 B is adapted from Vicini et al. (1998) with permission from the Journal of Neurophysiology. (C) Single-channel recordings of currents from outside-out membrane patches obtained from HEK293 cells expressing recombinant NMDA receptor subtypes. The single-channel recordings demonstrate distinct open probabilities and channel conductances depending on the GluN2 subunit in the diheteromeric NMDA receptor. Highlights of individual openings are shown on the left. Adapted from Yuan et al. (2008).

Figure 4.

Domain organization and ligand-binding sites in NMDA receptors. (A) The linear representation of the polypeptide chain illustrates the segments that form the four semiautonomous subunit domains shown in the cartoon, which are the extracellular ATD, the ABD, the TMD formed by three transmembrane helices (M1, M2, and M4) and a membrane reentrant loop (M2), and the intracellular CTD. The ABD is formed by two polypeptide segments (S1 and S2) that fold into a bilobed structure with an upper lobe (D1) and lower lobe (D2). The agonist-binding site is located in the cleft between the two lobes. (B) The crystal structure of the GluN1/2B NMDA receptor (Protein Data Bank accession no. 4PE5; Karakas and Furukawa, 2014) shows the subunit arrangement and the layered domain organization. The binding sites for agonists (and competitive antagonists) as well as predicted and known binding sites for PAMs and NAMs are highlighted. The figure is adapted from Hansen et al. (2017).

Figure 5.

Crystal structures of NMDA receptor ABDs. (A) Structures of the soluble GluN1/2 ABD heterodimers reveal the subunit interface and back-to-back dimer arrangement of the ABDs. The structure shown here is for the GluN1/2A ABD heterodimer with bound glutamate and glycine shown as spheres (Protein Data Bank accession no. 5I57; Yi et al., 2016). The top view of the structure highlights sites I–III at the subunit interface. (B) Overlay of crystal structures of GluN1/2A ABD heterodimers in complex with glycine and either glutamate agonist (Protein Data Bank accession no. 5I57; Yi et al., 2016) or a competitive glutamate site antagonist (Protein Data Bank accession no. 5U8C; Romero-Hernandez and Furukawa, 2017). Activation of NMDA receptors requires agonist-induced ABD closure. Competitive antagonists bind the ABD without inducing domain closure, thereby preventing receptor activation. (C) Magnified views of the glutamate-binding site with bound GluN2A-preferring antagonists NVP-AAM077 (Protein Data Bank accession no. 5U8C; Romero-Hernandez and Furukawa, 2017) or ST3 (Protein Data Bank accession no. 5VII; Lind et al., 2017). Schild analyses demonstrated that NVP-AAM077 has 11-fold and ST3 has 15-fold preference for GluN1/2A over GluN1/2B receptors (data adapted from Lind et al., 2017). The crystal structures reveal a binding mode in which NVP-AAM077 and ST3 occupy a cavity that extends toward GluN1 at the subunit interface, and mutational analyses show that the GluN2A preference of these antagonists is primarily mediated by four nonconserved residues (Lys738, Tyr754, Ile755, and Thr758) that do not directly contact the ligand but are positioned within 12 Å of the glutamate-binding site. (D) Structure of the agonist-bound GluN1/2A ABD heterodimer with the NAM MPX-007 bound at site II in the subunit interface (Protein Data Bank accession no. 5I59; Yi et al., 2016). (E) Magnified views of site II in GluN1/2A ABD heterodimer with bound MPX-007 (NAM; Protein Data Bank accession no. 5I59; Yi et al., 2016) or PAM GNE-8324 (Protein Data Bank accession no. 5H8Q; Hackos et al., 2016). The overlay illustrates the distinct effects of NAM and PAM binding on Val783 in GluN2A and Tyr535 in GluN1. The GluN2A selectivity of the NAMs and PAMs binding at this modulatory site is mediated by Val783 in GluN2A, which is nonconserved among GluN2 subunits (Phe in GluN2B and Leu in GluN2C/GluN2D).

Figure 6.

Structure of the intact NMDA receptors. (A) Structure of the glycine- and glutamate-bound GluN1-1b/2B NMDA receptor without CTDs (Protein Data Bank accession no. 5FXI; Tajima et al., 2016). (B) The GluN1 (1) and GluN2 (2) subunits are arranged as a dimer of heterodimers at the ATD and ABD layers in a 1-2-1-2 fashion. Note that the heterodimer pairs are interchanged between the ATD and ABD layers (i.e., subunit crossover). In the TMD layer, the GluN1 and GluN2 subunits are arranged as a tetramer with pseudo-fourfold symmetry. (C) Comparison of the two major conformational states observed in the presence of glycine and glutamate by cryo-EM/single-particle analysis. Shown in spheres are the Cα of the gating ring residues, GluN1-1b Arg684 and GluN2B Glu658, which are adjacent to the pore-forming M3 transmembrane helices. In the nonactive (Protein Data Bank accession no. 5FXI; Tajima et al., 2016) and active (Protein Data Bank accession no. 5FXG; Tajima et al., 2016) conformations, the distances between the two GluN2B Glu658 Cα atoms are ∼29 Å and ∼45 Å, respectively, indicating that degrees of tension in the ABD–TMD loops are different.

Figure 7.

Single-channel recordings of NMDA receptor gating. Recording of receptor activation (i.e., channel gating or pore dilation) in an excised outside-out membrane patch containing a single GluN1/2B receptor exposed to 1 mM glutamate plus 30 µM glycine for 1 ms as indicated. In this example, receptor activation results in a characteristically long burst of channel openings and closings (duration 128 ms). Evaluation of closed periods within the GluN1/2B activation suggests that two kinetically distinct pregating steps exist (i.e., fast and slow steps; see Fig. 8 D for model). Some (but not all) closures within the activation will reflect reversal of pore dilation, reversal of a single pregating step, followed by forward movement back through the pregating step and pore dilation. Two possible closures that might reflect the slow and fast pregating components are highlighted in red. Data are from Banke and Traynelis (2003).

Figure 8.

Application of a gating reaction mechanism of NMDA receptors. (A) Individual responses from a recombinant GluN1/2B channel in an excised outside-out patch activated by 1 ms application of maximally effective glutamate and glycine (indicated by the gray vertical bar and the open tip recording above the channel recordings). The patch contained a single active channel, which allowed analysis of the variable delay before channel opening. NMDA receptors bind agonist rapidly and subsequently open after a multimillisecond delay that reflects transition through kinetically distinct protein conformations before pore dilation (i.e., channel gating). Note that although application of maximal glutamate and glycine always produces a binding event, not all binding events lead to channel opening. Reproduced from Erreger et al. (2005a). (B) The cumulative plot of latency to opening after application of 1 mM glutamate for 1 ms. (C) The average of all individual recordings of single activations produced a macroscopic waveform with a characteristic rise time. (D) Evaluation of closed periods within the GluN1/2B activation suggested a model where two pregating steps can occur in any order and explosive opening of the pore, which occurs faster than the resolution of the recordings, is assumed to happen instantaneously once both pregating steps have been traversed. (E) Simulation of a single activation for a GluN1/2B channel (using the model in D) illustrates how brief gaps can contain information about forward rates for the fast kinetically distinct pregating step. The color above the simulation indicates occupancy in the corresponding closed state of the model in D. The slow step often reverses again through the fast state (green) before reopening.

Figure 9.

General pore structure of NMDA receptors. (A) Pore-lining elements contributed by the GluN1 subunit (blue; Protein Data Bank accession no. 5UN1; Song et al., 2018). The M3 transmembrane segment lines the extracellular part of the permeation pathway, whereas the M2 pore loop lines the intracellular part with the N site asparagine (red circle) positioned at the tip of the M2 pore loop. The channel is in the closed conformation. (B) The narrow constriction is formed by nonhomologous asparagine residues, the GluN1 N site and the GluN2 N+1 site (Wollmuth et al., 1996; Song et al., 2018). The GluN2B subunit is colored orange. For both GluN1 and GluN2, the N site asparagine residue is positioned at the tip of the M2 loop.