Subunit Arrangement and Phenylethanolamine Binding in GluN1/GluN2B NMDA Receptors

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Summary

Since it was unexpectedly discovered that the anti-hypertensive agent, ifenprodil, has neuroprotective activity through effects to N-methyl-D-aspartate (NMDA) receptors ¹, enormous efforts have been made to understand the mechanism of action and to develop improved therapeutic compounds based on this knowledge ^{2–
4}. Neurotransmission mediated by NMDA receptors is essential for basic brain development and function ⁵. These receptors form heteromeric ion channels and become activated upon concurrent binding of glycine and glutamate to the GluN1 and GluN2 subunits, respectively. A functional hallmark of NMDA receptors is that their ion channel activity is allosterically regulated by binding of small compounds to the amino terminal domain (ATD) in a subtype specific manner. Ifenprodil and related phenylethanolamine compounds, which specifically inhibit GluN1/GluN2B NMDA receptors ^{6,
7}, have been intensely studied for their potential use in treatment of various neurological disorders and diseases including depression, Alzheimer’s disease and Parkinson’s disease ^{2,
4}. Despite great enthusiasm, mechanisms underlying recognition of phenylethanolamines and the ATD-mediated allosteric inhibition remain limited due to lack of structural information. Here we report that the GluN1 and GluN2B ATDs form heterodimer and that phenylethanolamine binds at the GluN1-GluN2B subunit interface rather than within the GluN2B cleft. The crystal structure of the GluN1b/GluN2B ATD heterodimer shows a highly distinct pattern of subunit arrangement that is different from those observed in homodimeric non-NMDA receptors and reveals the molecular determinants for phenylethanolamine binding. Restriction of domain movement in the bi-lobed structures of the GluN2B ATD by engineering an inter-subunit disulfide bond dramatically decreases ifenprodil-sensitivity indicating that conformational freedom in the GluN2B ATD is essential for ifenprodil-mediated allosteric inhibition in NMDA receptors.

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Most cited references 22

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Subunit arrangement and function in NMDA receptors.

Hiroyasu Furukawa, Satinder K Singh, Romina Mancusso … (2005)

Excitatory neurotransmission mediated by NMDA (N-methyl-D-aspartate) receptors is fundamental to the physiology of the mammalian central nervous system. These receptors are heteromeric ion channels that for activation require binding of glycine and glutamate to the NR1 and NR2 subunits, respectively. NMDA receptor function is characterized by slow channel opening and deactivation, and the resulting influx of cations initiates signal transduction cascades that are crucial to higher functions including learning and memory. Here we report crystal structures of the ligand-binding core of NR2A with glutamate and that of the NR1-NR2A heterodimer with glutamate and glycine. The NR2A-glutamate complex defines the determinants of glutamate and NMDA recognition, and the NR1-NR2A heterodimer suggests a mechanism for ligand-induced ion channel opening. Analysis of the heterodimer interface, together with biochemical and electrophysiological experiments, confirms that the NR1-NR2A heterodimer is the functional unit in tetrameric NMDA receptors and that tyrosine 535 of NR1, located in the subunit interface, modulates the rate of ion channel deactivation.

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Protein complex expression by using multigene baculoviral vectors.

Stewart Richmond, Philipp Berger, John FitzGerald … (2006)

Elucidation of the molecular basis of protein-interaction networks, in particular in higher eukaryotes, is hampered by insufficient quantities of endogenous multiprotein complexes. Present recombinant expression methods often require considerable investment in both labor and materials before multiprotein expression, and after expression and biochemical analysis these methods do not provide flexibility for expressing an altered multiprotein complex. To meet these demands, we have recently introduced MultiBac, a modular baculovirus-based system specifically designed for eukaryotic multiprotein expression. Here we describe new transfer vectors and a combination of DNA recombination-based methods, which further facilitate the generation of multigene cassettes for protein coexpression (Fig. 1), thus providing a flexible platform for generation of protein expression vectors and their rapid regeneration for revised expression studies. Genes encoding components of a multiprotein complex are inserted into a suite of compatible transfer vectors by homologous recombination. These progenitor constructs are then rapidly joined in the desired combination by Cre-loxP-mediated in vitro plasmid fusion. Protocols for integration of the resulting multigene expression cassettes into the MultiBac baculoviral genome are provided that rely on Tn7 transposition and/or Cre-loxP reaction carried out in vivo in Escherichia coli cells tailored for this purpose. Detailed guidelines for multigene virus generation and amplification, cell culture maintenance and protein production are provided, together with data illustrating the simplicity and remarkable robustness of the present method for multiprotein expression using a composite MultiBac baculoviral vector.

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Allosteric modulators of NR2B-containing NMDA receptors: molecular mechanisms and therapeutic potential.

Martin J. Gunthorpe, Pierre Paoletti, Laetitia Mony … (2009)

N-methyl-D-aspartate receptors (NMDARs) are ion channels gated by glutamate, the major excitatory neurotransmitter in the mammalian central nervous system (CNS). They are widespread in the CNS and are involved in numerous physiological and pathological processes including synaptic plasticity, chronic pain and psychosis. Aberrant NMDAR activity also plays an important role in the neuronal loss associated with ischaemic insults and major degenerative disorders including Parkinson's and Alzheimer's disease. Agents that target and alter NMDAR function may, thus, have therapeutic benefit. Interestingly, NMDARs are endowed with multiple extracellular regulatory sites that recognize ions or small molecule ligands, some of which are likely to regulate receptor function in vivo. These allosteric sites, which differ from agonist-binding and channel-permeation sites, provide means to modulate, either positively or negatively, NMDAR activity. The present review focuses on allosteric modulation of NMDARs containing the NR2B subunit. Indeed, the NR2B subunit confers a particularly rich pharmacology with distinct recognition sites for exogenous and endogenous allosteric ligands. Moreover, NR2B-containing receptors, compared with other NMDAR subtypes, appear to contribute preferentially to pathological processes linked to overexcitation of glutamatergic pathways. The actions of extracellular H+, Mg2+, Zn2+, of polyamines and neurosteroids, and of the synthetic compounds ifenprodil and derivatives ('prodils') are presented. Particular emphasis is put upon the structural determinants and molecular mechanisms that underlie the effects exerted by these agents. A better understanding of how NR2B-containing NMDARs (and NMDARs in general) operate and how they can be modulated should help define new strategies to counteract the deleterious effects of dysregulated NMDAR activity.

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Author and article information

Journal

Journal ID (nlm-journal-id): 0410462

Journal ID (pubmed-jr-id): 6011

Journal ID (nlm-ta): Nature

Journal ID (iso-abbrev): Nature

Title: Nature

ISSN (Print): 0028-0836

ISSN (Electronic): 1476-4687

Publication date Nihms-submitted: 21 July 2011

Publication date (Electronic): 15 June 2011

Publication date PMC-release: 14 January 2012

Volume: 475

Issue: 7355

Pages: 249-253

Affiliations

[1 ]Cold Spring Harbor Laboratory, WM Keck Structural Biology Laboratory 1 Bungtown Road, Cold Spring Harbor, NY 11724

Author notes

[* ]To whom correspondence should be addressed Hiro Furukawa, Cold Spring Harbor Laboratory, WM Keck Structural Biology Laboratory, 1 Bungtown Rd., Cold Spring Harbor, NY 11724, U.S.A. Tel: 1-516-367-8872; Fax: 1-516-367-8873; furukawa@ 123456cshl.edu

Article

Manuscript ID: nihpa313083

DOI: 10.1038/nature10180

PMC ID: 3171209

PubMed ID: 21677647

SO-VID: 4a5b2487-4293-4c58-87a4-6d6492fa1ec8

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History

Funding

Funded by: National Institute of Mental Health : NIMH

Award ID: R01 MH085926-03 || MH

Funded by: National Institute of Mental Health : NIMH

Award ID: R01 MH085926-02 || MH

Funded by: National Institute of Mental Health : NIMH

Award ID: R01 MH085926-01A1 || MH

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