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NMDA receptor structures reveal subunit arrangement and pore architecture.

Lee CH, Lü W, Michel JC, Goehring A, Du J, Song X, Gouaux E - Nature (2014)

Bottom Line: Receptor subunits are arranged in a 1-2-1-2 fashion, demonstrating extensive interactions between the amino-terminal and ligand-binding domains.The transmembrane domains harbour a closed-blocked ion channel, a pyramidal central vestibule lined by residues implicated in binding ion channel blockers and magnesium, and a ∼twofold symmetric arrangement of ion channel pore loops.These structures provide new insights into the architecture, allosteric coupling and ion channel function of NMDA receptors.

View Article: PubMed Central - PubMed

Affiliation: 1] Vollum Institute, Oregon Health & Science University, 3181 SW Sam Jackson Park Road, Portland, Oregon 97239, USA [2].

ABSTRACT
N-methyl-d-aspartate (NMDA) receptors are Hebbian-like coincidence detectors, requiring binding of glycine and glutamate in combination with the relief of voltage-dependent magnesium block to open an ion conductive pore across the membrane bilayer. Despite the importance of the NMDA receptor in the development and function of the brain, a molecular structure of an intact receptor has remained elusive. Here we present X-ray crystal structures of the Xenopus laevis GluN1-GluN2B NMDA receptor with the allosteric inhibitor, Ro25-6981, partial agonists and the ion channel blocker, MK-801. Receptor subunits are arranged in a 1-2-1-2 fashion, demonstrating extensive interactions between the amino-terminal and ligand-binding domains. The transmembrane domains harbour a closed-blocked ion channel, a pyramidal central vestibule lined by residues implicated in binding ion channel blockers and magnesium, and a ∼twofold symmetric arrangement of ion channel pore loops. These structures provide new insights into the architecture, allosteric coupling and ion channel function of NMDA receptors.

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Electrophysiology and Western analysis of GluN1 Δ/GluN2B Δ receptor combinationsa, b, c, Representative TEVC currents recorded for oocytes expressing GluN1 Δ4 and  (a) GluN2B Δ1 or  (b, c) GluN2B Δ3 receptors in response to agonist (100µM glycine and 100 µM glutamate, bars, 20 sec) or agonist plus 1 mM MgCl2 (indicated) after soaking oocytes in the  (a, b) absence or  (c) presence of 5mM DTT. d, Western blot analysis of oocytes demonstrating spontaneously crosslinking cysteines (Lys216Cys) introduced at the GluN2B Δ3 intersubunit interface. Oocytes were soaked in the absence (left lanes) or presence of 5mM DTT (right lanes) before processing for Western analysis using an anti-GluN2B antibody. Filled and open triangles indicate positions of crosslinked and monomeric GluN2B, respectively. e, Graph of mean agonist-induced inward currents from four reduced oocytes expressing GluN1 Δ4 and GluN2B Δ3 in the absence (G/G, −25 ± −4 nA) or presence of 1mM MgCl2 (G/G/Mg2+, 8 ± 5 nA). Error bars represent s.e.m. The p value is <0.001 for the paired T-test (asterisk). f, Representative TEVC currents recorded in response to agonist (100µM glycine and 100 µM glutamate bars, 10 sec) or agonist plus 1 mM MgCl2 for oocytes expressing constructs similar to the GluN1 Δ2/GluN2B Δ2 receptor combination with the following exceptions: GluN1 subunit, Asp656 (wt), Gly636Arg and Lys741Asp; and GluN2B subunit, Glu654 (wt), Glu655 (wt), and Lys216 (wt). g, Binding constants for the GluN1 Δ2/GluN2B Δ2 construct.
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Figure 8: Electrophysiology and Western analysis of GluN1 Δ/GluN2B Δ receptor combinationsa, b, c, Representative TEVC currents recorded for oocytes expressing GluN1 Δ4 and (a) GluN2B Δ1 or (b, c) GluN2B Δ3 receptors in response to agonist (100µM glycine and 100 µM glutamate, bars, 20 sec) or agonist plus 1 mM MgCl2 (indicated) after soaking oocytes in the (a, b) absence or (c) presence of 5mM DTT. d, Western blot analysis of oocytes demonstrating spontaneously crosslinking cysteines (Lys216Cys) introduced at the GluN2B Δ3 intersubunit interface. Oocytes were soaked in the absence (left lanes) or presence of 5mM DTT (right lanes) before processing for Western analysis using an anti-GluN2B antibody. Filled and open triangles indicate positions of crosslinked and monomeric GluN2B, respectively. e, Graph of mean agonist-induced inward currents from four reduced oocytes expressing GluN1 Δ4 and GluN2B Δ3 in the absence (G/G, −25 ± −4 nA) or presence of 1mM MgCl2 (G/G/Mg2+, 8 ± 5 nA). Error bars represent s.e.m. The p value is <0.001 for the paired T-test (asterisk). f, Representative TEVC currents recorded in response to agonist (100µM glycine and 100 µM glutamate bars, 10 sec) or agonist plus 1 mM MgCl2 for oocytes expressing constructs similar to the GluN1 Δ2/GluN2B Δ2 receptor combination with the following exceptions: GluN1 subunit, Asp656 (wt), Gly636Arg and Lys741Asp; and GluN2B subunit, Glu654 (wt), Glu655 (wt), and Lys216 (wt). g, Binding constants for the GluN1 Δ2/GluN2B Δ2 construct.

Mentions: The first structure of the NMDA receptor was derived from the low resolution Data set 4 (Extended Data Table 2) and involved a construct lacking the GluN2B K216C mutant. In this crystal form, there are two halves of a receptor in the asymmetric unit and application of crystal symmetry creates two intact receptors, each with a different conformation of the ATDs in which the angles of the ATD domains range from 59° to 84° across the overall 2-fold axis (Fig. 2d). We further observed that helix α5 of the GluN2B R2 lobes face each other, proximal to the overall 2-fold axis of symmetry. Because we hypothesized that these structures were indicative of substantial mobility in the ATD layer, we made single cysteine substitutions on the exposed face of helix α5 and screened for redox dependent cross linking of GluN2B subunits. Indeed, the K216C mutant, as well as other residues on the face of α5, spontaneously form subunit-subunit cross links (Extended Data Figs. 1, 2, 4), bringing the GluN2B ATDs in close apposition (Extended Data Fig. 5e), diminishing ion channel activity and increasing the resolution to which the crystals diffract. In two electrode voltage clamp experiments, reduction of oocytes using dithiothreitol enhances current responses from the K216C mutant, suggesting that movements of the ATDs allosterically modulate the activity of the ion channel (Extended Data Fig. 2).


NMDA receptor structures reveal subunit arrangement and pore architecture.

Lee CH, Lü W, Michel JC, Goehring A, Du J, Song X, Gouaux E - Nature (2014)

Electrophysiology and Western analysis of GluN1 Δ/GluN2B Δ receptor combinationsa, b, c, Representative TEVC currents recorded for oocytes expressing GluN1 Δ4 and  (a) GluN2B Δ1 or  (b, c) GluN2B Δ3 receptors in response to agonist (100µM glycine and 100 µM glutamate, bars, 20 sec) or agonist plus 1 mM MgCl2 (indicated) after soaking oocytes in the  (a, b) absence or  (c) presence of 5mM DTT. d, Western blot analysis of oocytes demonstrating spontaneously crosslinking cysteines (Lys216Cys) introduced at the GluN2B Δ3 intersubunit interface. Oocytes were soaked in the absence (left lanes) or presence of 5mM DTT (right lanes) before processing for Western analysis using an anti-GluN2B antibody. Filled and open triangles indicate positions of crosslinked and monomeric GluN2B, respectively. e, Graph of mean agonist-induced inward currents from four reduced oocytes expressing GluN1 Δ4 and GluN2B Δ3 in the absence (G/G, −25 ± −4 nA) or presence of 1mM MgCl2 (G/G/Mg2+, 8 ± 5 nA). Error bars represent s.e.m. The p value is <0.001 for the paired T-test (asterisk). f, Representative TEVC currents recorded in response to agonist (100µM glycine and 100 µM glutamate bars, 10 sec) or agonist plus 1 mM MgCl2 for oocytes expressing constructs similar to the GluN1 Δ2/GluN2B Δ2 receptor combination with the following exceptions: GluN1 subunit, Asp656 (wt), Gly636Arg and Lys741Asp; and GluN2B subunit, Glu654 (wt), Glu655 (wt), and Lys216 (wt). g, Binding constants for the GluN1 Δ2/GluN2B Δ2 construct.
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Figure 8: Electrophysiology and Western analysis of GluN1 Δ/GluN2B Δ receptor combinationsa, b, c, Representative TEVC currents recorded for oocytes expressing GluN1 Δ4 and (a) GluN2B Δ1 or (b, c) GluN2B Δ3 receptors in response to agonist (100µM glycine and 100 µM glutamate, bars, 20 sec) or agonist plus 1 mM MgCl2 (indicated) after soaking oocytes in the (a, b) absence or (c) presence of 5mM DTT. d, Western blot analysis of oocytes demonstrating spontaneously crosslinking cysteines (Lys216Cys) introduced at the GluN2B Δ3 intersubunit interface. Oocytes were soaked in the absence (left lanes) or presence of 5mM DTT (right lanes) before processing for Western analysis using an anti-GluN2B antibody. Filled and open triangles indicate positions of crosslinked and monomeric GluN2B, respectively. e, Graph of mean agonist-induced inward currents from four reduced oocytes expressing GluN1 Δ4 and GluN2B Δ3 in the absence (G/G, −25 ± −4 nA) or presence of 1mM MgCl2 (G/G/Mg2+, 8 ± 5 nA). Error bars represent s.e.m. The p value is <0.001 for the paired T-test (asterisk). f, Representative TEVC currents recorded in response to agonist (100µM glycine and 100 µM glutamate bars, 10 sec) or agonist plus 1 mM MgCl2 for oocytes expressing constructs similar to the GluN1 Δ2/GluN2B Δ2 receptor combination with the following exceptions: GluN1 subunit, Asp656 (wt), Gly636Arg and Lys741Asp; and GluN2B subunit, Glu654 (wt), Glu655 (wt), and Lys216 (wt). g, Binding constants for the GluN1 Δ2/GluN2B Δ2 construct.
Mentions: The first structure of the NMDA receptor was derived from the low resolution Data set 4 (Extended Data Table 2) and involved a construct lacking the GluN2B K216C mutant. In this crystal form, there are two halves of a receptor in the asymmetric unit and application of crystal symmetry creates two intact receptors, each with a different conformation of the ATDs in which the angles of the ATD domains range from 59° to 84° across the overall 2-fold axis (Fig. 2d). We further observed that helix α5 of the GluN2B R2 lobes face each other, proximal to the overall 2-fold axis of symmetry. Because we hypothesized that these structures were indicative of substantial mobility in the ATD layer, we made single cysteine substitutions on the exposed face of helix α5 and screened for redox dependent cross linking of GluN2B subunits. Indeed, the K216C mutant, as well as other residues on the face of α5, spontaneously form subunit-subunit cross links (Extended Data Figs. 1, 2, 4), bringing the GluN2B ATDs in close apposition (Extended Data Fig. 5e), diminishing ion channel activity and increasing the resolution to which the crystals diffract. In two electrode voltage clamp experiments, reduction of oocytes using dithiothreitol enhances current responses from the K216C mutant, suggesting that movements of the ATDs allosterically modulate the activity of the ion channel (Extended Data Fig. 2).

Bottom Line: Receptor subunits are arranged in a 1-2-1-2 fashion, demonstrating extensive interactions between the amino-terminal and ligand-binding domains.The transmembrane domains harbour a closed-blocked ion channel, a pyramidal central vestibule lined by residues implicated in binding ion channel blockers and magnesium, and a ∼twofold symmetric arrangement of ion channel pore loops.These structures provide new insights into the architecture, allosteric coupling and ion channel function of NMDA receptors.

View Article: PubMed Central - PubMed

Affiliation: 1] Vollum Institute, Oregon Health & Science University, 3181 SW Sam Jackson Park Road, Portland, Oregon 97239, USA [2].

ABSTRACT
N-methyl-d-aspartate (NMDA) receptors are Hebbian-like coincidence detectors, requiring binding of glycine and glutamate in combination with the relief of voltage-dependent magnesium block to open an ion conductive pore across the membrane bilayer. Despite the importance of the NMDA receptor in the development and function of the brain, a molecular structure of an intact receptor has remained elusive. Here we present X-ray crystal structures of the Xenopus laevis GluN1-GluN2B NMDA receptor with the allosteric inhibitor, Ro25-6981, partial agonists and the ion channel blocker, MK-801. Receptor subunits are arranged in a 1-2-1-2 fashion, demonstrating extensive interactions between the amino-terminal and ligand-binding domains. The transmembrane domains harbour a closed-blocked ion channel, a pyramidal central vestibule lined by residues implicated in binding ion channel blockers and magnesium, and a ∼twofold symmetric arrangement of ion channel pore loops. These structures provide new insights into the architecture, allosteric coupling and ion channel function of NMDA receptors.

Show MeSH
Related in: MedlinePlus