From bedside-to-bench: What disease-associated variants are teaching us about the NMDA receptor

Abstract

NMDA receptors (NMDARs) are glutamate-gated ion channels that contribute to nearly all brain processes. Not surprisingly then, genetic variations in the genes encoding NMDAR subunits can be associated with neurodevelopmental, neurological and psychiatric disorders. These disease-associated variants (DAVs) present challenges, such as defining how DAV-induced alterations in receptor function contribute to disease progression and how to treat the affected individual clinically. As a starting point to overcome these challenges, we need to refine our understanding of the complexity of NMDAR structure function. In this regard, DAVs have expanded our knowledge of NMDARs because they do not just target well-known structure-function motifs, but rather give an unbiased view of structural elements that are important to the biology of NMDARs. Indeed, established NMDAR structure-function motifs have been validated by the appearance of disorders in patients where these motifs have been altered, and DAVs have identified novel structural features in NMDARs such as gating triads and hinges in the gating machinery. Still, the majority of DAVs remain unexplored and occur at sites in the protein with unidentified function or alter receptor properties in multiple and unanticipated ways. Detailed mechanistic and structural investigations are required of both established and novel motifs to develop a highly refined pathomechanistic model that accounts for the complex machinery that regulates NMDARs. Such a model would provide a template for rational drug design and a starting point for personalized medicine.

Keywords: C-terminal domain; GRIN1; GRIN2A; GRIN2B; GRIN2C; GRIN2D; NMDA receptor; amino-terminal domain; gating hinge; ligand-binding domain; pre-active; pre-gating; precision medicine; transmembrane domain.

Figure 1.. Disease-associated variants (DAV) are prominent in the core gating machinery in NMDA receptors (NMDAR).

(A) Membrane topology of the tetrameric NMDAR, which are composed of two GluN1 (= A/C conformation = light orange) and two GluN2 (= B/D conformation = gray) subunits. NMDARs are highly modular proteins composed of 4 layered domains: extracellular amino-terminal (ATD) and ligand-binding (LBD) domains, a membrane embedded transmembrane domain (TMD) forming the ion channel, and an intracellular C-terminal domain (CTD). The LBD and TMD are connected by short polypeptide chains called the LBD-TMD linkers. Model structure based on 4TLM of GluN1/GluN2B (Amin et al., 2017). (B) Percent and number (in parenthesis) of unique positions containing a DAV, relative to total number of residues, within each structural layer for GluN1 (GRIN1), GluN2A (GRIN2A), and GluN2B (GRIN2B). DAVs are also present in GluN2C (Yu et al., 2018) and GluN2D (XiangWei et al., 2019) subunits, but are too few in number to include in this analysis. Unique DAVs are missense mutations only, excluding terminations and deletions, and do not include different variations at the same position (Table 2). (C) Topology of an individual subunit (GluN1) highlighting that the modular domains are intrinsic to subunits. The various structural levels are colored: ATD (blue), LBD (green), LBD-TMD linkers (red), TMD (orange), and CTD (light gray). (D) Cartoon of the core gating machinery in NMDARs transitioning from the agonist unbound, ion channel closed (left) to the agonist bound, ion channel open (right) conformation. Binding of agonists to the LBD (green) causes the LBD clamshell, formed by D1 and D2, to close (transition from left to right). The movement of D2 away from the membrane pulls on the M3-S2 linker which in turn pulls on the activation gate formed at the apex of M3 leading to ion channel opening (transition from left to right). Open NMDARs permeates monovalent cations (Na⁺) and Ca²⁺. Below the cartoons are high resolution structures of the AMPAR M3 helices from a top down view as the channel transitions from closed (left) to open (right). PDBs 4WEK & 4WEO (Twomey et al., 2017). At present, open state structures are only available for AMPARs.

Figure 2.. DAVs in the ligand-binding domains (LBD) have diverse functional effects.

(A) Cartoon of agonist-bound core gating machinery. (B) Location of DAVs in the agonist-bound GluN1 (left), GluN2A (middle), and GluN2B (right) LBDs. The D1 and D2 lobes are highlighted and the ligand binding pockets are circled. DAVs are in red. PDBs 5I57 (Yi et al., 2016) & 4PE5 (Karakas & Furukawa, 2014). (C) Zoomed in view of the ligand binding pocket for GluN1 (left), GluN2A (center), and GluN2B (right). The molecular structure of agonists (glycine, GluN1; glutamate, GluN2 subunits) are indicated. DAVs located in the vicinity of the binding pockets are indicated in green and are labeled. (D) Venn diagrams showing the functional effects of DAVs tested from the GluN1 (left), GluN2A (middle), and GluN2B (right) LBDs (Fry et al., 2018).

Figure 3.. DAVs in the M3-S2 linkers and major pore lining structures.

(A) View of GluN1 (light orange) and GluN2B (gray) M3-S2 linkers and pore forming structures. The iGluR pore is formed by the four M3 transmembrane helices and on the intracellular half, by the four M2 pore loops. The M3 segments contain SYTANLAAF, the most highly conserved motif in iGluRs. At their apex, the M3 segments form an activation gate, which prevents the flux of ions in the closed state. The M2 pore loop forms the narrow constriction or selectivity filter. (B) View of the GluN1 pore lining elements with DAVs highlighted in hot pink. The location of the SYTANLAAF motif is indicated in cyan. (C) View of the GluN2 pore lining elements with DAVs in blue (GluN2A), red (GluN2B), or purple (both GluN2A & GluN2B). (D) View of M2 pore loops with the nearest pore loop removed for clarity. The GluN1 N site and the GluN2 N+1 site form the selectivity filter (Wollmuth et al., 1996) and play key roles in Ca²⁺ permeability and Mg²⁺ block (Traynelis et al., 2010). PDB 5UN1 (Song et al., 2018). (E) Sequence of GluN1, GluN2A, and GluN2B M2 pore loops highlighting secondary structures and positions of N and N+1 sites (Song et al., 2018). Location of DAVs are indicated in red.

Figure 4.. DAVs in the outer structures, M1 and M4, identify key structure-function motifs.

(A) Top down view of the TMD. The inner structures, the M2 loops (not shown) and M3 helices, form the ion channel pore (solid circle). The outer structures, M1 and M4 helices and the linkers that connect them to the LBD, S1-M1 and S2-M4, surround the inner core. Together, the S1-M1 (notably the pre-M1 helix), extracellular M3, and extracellular M4, make up a structure-function motif critical to gating known as the gating triad (red circles and dashed lines). The gating triad is present in both the GluN1 and GluN2 subunits though it has most notable effects in GluN2 (Ogden et al., 2017). (B) Cartoon of the inner and outer structures showing the general displacement of structures in the transition from the closed (left) to open (right) conformation. (C) Topology of the outer structures, the M1 helices and their LBD-TMD linker (S1-M1) (left) and the M4 helices and their LBD-TMD linker (S2-M4) (right) for GluN1 and GluN2. The extracellular portion of the TMD and the lower portion of the LBD-TMD linkers are termed the gating collar (Yelshanskaya et al., 2017) or gating triad (Gibb et al., 2018). DAVs are highlighted in hot pink (GluN1), blue (GluN2A), red (GluN2B), or purple (both GluN2A & GluN2B). The helices are oriented such that the M3 facing portion is to the left. (D) The three-dimensional arrangement of the gating triad in the closed state. The GluN2 pre-M1 helix, extracellular M4 and M3 make up the gating triad. With the exception of the M2 pore loop and pore lining portions of M3, most DAVs in the TMD appear in this region. DAVs are labeled as in (C).

Figure 5.. Agonist-induced displacements of the outer structures prime the ion channel for pore opening.

Top down views of the gating triad in AMPARs for the B/D (≈GluN2) subunit. In the transition from the closed (left) to the open (right) states, all three elements – the pre-M1 helix, and the tops of M4 and M3 – undergo extensive and coordinated movements. The splaying of the M3 segments, highlighted in red (right), is the final step in ion channel opening. It seems likely that the outer structures undergo displacements (pre-M1, blue & M4, green) (middle panel) prior to the final M3 displacement. These displacement between agonist binding and M3-mediated pore opening are called pre-active or pre-gating movements.

Figure 6.. DAVs identify critical gating hinges.

(A) Top down view of the NMDAR TMD in the closed state with M1 omitted for clarity (left) and a simulated open state (right) (Amin et al., 2018). There are multiple hinge points including an alanine hinge in the M3 SYTANLAAF motif (discussed with M3 segment) and a glycine hinge (conserved G) in the M4 segments (hinges labeled in cyan). These hinges facilitate the transition to the open state (red arrows). (B) Constrained (left) and expanded (right) conformations of the GluN1 M4 helix and the M2 pore loop. The lateral expansion of the GluN1 M4 at the conserved G hinge allows the M2 loop to expand, permitting the transition from a low (left) to high (right) Ca²⁺ permeability state. DAVs at the GluN1 conserved G keep the M4 segments in the constrained conformation (left). (C) Cartoon representation of the pore in two conformations: Unstable open (left) where the M3 gate is open but the GluN1 M4 is constrained; and Stable open (right) where both the M3 gate is open and GluN1 M4 is expanded. In the ‘unstable open’ conformation (left), NMDARs show brief ion channel openings at the single channel level (lower traces) and low Ca²⁺ permeability. In the ‘stable open’ conformation (right), NMDARs show more characteristic long-lived single channel openings (lower traces) and high Ca²⁺ permeability (Amin et al., 2018).

Figure 7.. DAVs in the modulatory amino-terminal (ATD) and C-terminal (CTD) domains.

(A) Location of DAVs in the GluN1 (left), GluN2A (middle), and GluN2B (right) ATDs. The R1 and R2 lobes are highlighted. Only GluN2A binds the inhibitory divalent zinc under physiological conditions. DAVs are in red. PDBs 5TQ2 & 5TPZ (Romero-Hernandez et al., 2016). (B) Linear representation of the GluN1, GluN2A, and GluN2B CTDs. At present, there are no high-resolution structures of the CTDs. Sites for known phosphorylation (Src kinase, Protein kinase C (PKC), Protein kinase A (PKA), palmitoylation, and putative phosphorylation (yellow) are indicated along with essential protein-protein interactions. DAVs are indicated by red bars. The vast majority of these DAVs reside at positions of unknown function.