A genetically modified mouse model probing the selective action of ifenprodil at the N-methyl-D-aspartate type 2B receptor
Merck Sharp and Dohme Research Laboratories, The Neuroscience Research Centre, Terlings Park, Eastwick Road, Harlow, Essex CM20 2QR, UK.Molecular and Cellular Neuroscience (Impact Factor: 3.84). 10/2006; 33(1):47-56. DOI: 10.1016/j.mcn.2006.06.006
Selective antagonism of N-methyl-d-aspartate (NMDA) 2B subunit containing receptors has been suggested to have potential therapeutic application for multiple CNS disorders. The amino terminal NR2B residues 1 to 282 were found to be both necessary and sufficient for the binding and function of highly NR2B subunit specific antagonists like ifenprodil and CP-101,606. Using a genetic approach in mice, we successfully replaced the murine NR2B gene function by "knocking-in" (KI) a chimeric human NR2A/B cDNA containing the minimal domain abolishing ifenprodil binding into the endogenous NR2B locus. Patch-clamp recording from hippocampal cultures of the NR2B KI mice demonstrated that their NMDA receptors have reduced sensitivity to both ifenprodil and CP-101,606, as predicted, but also have a lower affinity for glycine. The NR2B KI mice exhibited normal locomotor activity making this ifenprodil-insensitive mouse model a valuable tool to test the specificity of NR2B selective antagonists in vivo.
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- "Text boxes are used with citation numbers to indicate gain (no box) or loss (box) of function of the related property with the presence of the motif. Citations numbers are defined as follows: (1) Meddows et al., 2001, (2) Mullasseril et al., 2010, (3) Gielen et al., 2009, (4) Yuan et al., 2009, (5) Qiu et al., 2009, (6) Cousins et al., 2008, (7) Paoletti et al., 2000, (8) Cao et al., 2011, (9) Ng et al., 2008, (10) Rosahl et al., 2006, (11) Karakas et al., 2011, (12) Perin-Dureau et al., 2002, (13) Paoletti and Neyton, 2007, (14) Kalbaugh et al., 2004, (15) Chen et al., 2005, (16) Laube et al., 1997, (17) Anson et al., 1998, (18) Barria and Malinow, 2002, (19) Prybylowski et al., 2005, (20) Meguro et al., 1992, (21) Kuner and Schoepfer, 1996, (22) Horak et al., 2008, (23) Chang and Kuo, 2008, (24) Xu et al., 2012, (25) Talukder and Wollmuth, 2011, "
ABSTRACT: The numbers and types of ionotropic glutamate receptors at most vertebrate central excitatory synapses are altered as a function of changes in input activity patterns that occur during postnatal development. Activity-dependent developmental alterations in glutamate receptors underlie lasting changes in synaptic efficacy (plasticity) and metaplasticity (the plasticity of synaptic plasticity), which are critical elements of normal brain maturation. Understanding the specific involvement of glutamate receptors in synaptic development and function is made multiplicatively complex by the existence of a large number of glutamate receptor subunits, numerous subunit-specific amino acid sequences that regulate receptor function, and subunit-specific synaptic insertion restrictions imposed by associated anchoring proteins. Many receptor properties are altered when subunits are switched, so it is unclear which individual receptor property or properties underlie changes in synaptic function and plasticity during postnatal development. As a result, a more detailed understanding of the factors that regulate synaptic and cognitive development will involve mutations in glutamate receptor subunits that separate individual receptor properties and permit synaptic insertion at both immature and mature synapses in genetically modified organisms. This position paper focuses on structural modifications in N-methyl-d-aspartate receptors (NMDARs) that occur during postnatal forebrain development and attempts to provide a method for pursuing a more complete understanding of the functional ramifications of developmental alterations in NMDAR subunit composition.
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- "In contrast, there is no crystal structure for the putative allosteric modulators' binding sites located on the amino terminal domain proximal to the S1 region of ionotropic glutamate receptors, including NMDA receptors. The apparent high affinity Zn2+ binding site is situated in ATD of NR2A [20-22]; the putative phenylethanolamines binding site(s) in ATD of NR2B [23-25]; the polyamine-sensitive region in ATD of NR1 [26,27]; and possibly a Zn2+ binding site in NR2B . Indeed, Rosahl and colleagues  provided ex vivo support for the modulatory role of ATD of NR2B by ifenprodil and ifenprodil-like NMDA receptor antagonists recently. "
ABSTRACT: Phenylethanolamines selectively bind to NR2B subunit-containing N-methyl-D-aspartate-subtype of ionotropic glutamate receptors and negatively modulate receptor activity. To investigate the structural and functional properties of the ifenprodil binding domain on the NR2B protein, we have purified a soluble recombinant rat NR2B protein fragment comprising the first ~400 amino acid amino-terminal domain (ATD2B) expressed in E. coli. Spectral measurements on refolded ATD2B protein demonstrated specific binding to ifenprodil. We have used site-directed mutagenesis, circular dichroism spectroscopy and molecular modeling to obtain structural information on the interactions between critical amino acid residues and ifenprodil of our soluble refolded ATD2B proteins. Ligand-induced changes in protein structure were inferred from changes in the circular dichroism spectrum, and the concentration dependence of these changes was used to determine binding constants for ifenprodil and its analogues. Ligand binding of ifenprodil, RO25,6981 and haloperidol on soluble recombinant ATD2B determined from circular dichroism spectroscopy yielded low-to-high micromolar equilibrium constants which concurred with functional IC₅₀ measurement determined in heterologously expressed NR1/NR2B receptors in Xenopus oocytes. Amino acid residue substitutions of Asp101, Ile150 and Phe176 with alanine residue within the ATD2B protein altered the recombinant protein dissociation constants for ifenprodil, mirroring the pattern of their functional phenotypes. Molecular modeling of ATD2B as a clam-shell-like structure places these critical residues near a putative ligand binding site. We report for the first time biochemical measurements show that the functional measurements actually reflect binding to the ATD of NR2B subunit. Insights gained from this study help advance the theory that ifenprodil is a ligand for the ATD of NR2B subunit.
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ABSTRACT: The amino-terminal domains (ATDs) of N-methyl-d-aspartate (NMDA) receptors contain binding sites for modulators and may serve as potential drug targets in neurological diseases. Here, three fusion tags (6xHis-, GST-, and MBP-) were fused to the ATD of NMDA receptor NR2B subunit (ATD2B) and expressed in Escherichia coli. Each tag's ability to confer enhanced solubility to ATD2B was assessed. Soluble ATD2B was successfully obtained as a MBP fusion protein. Dynamic light scattering revealed the protein (1mg/ml) exists as monodispersed species at 25 degrees C. Functional studies using circular dichroism showed that the soluble MBP-ATD2B bound ifenprodil in a dose-dependent manner. The dissociation constants obtained for ifenprodil were similar in the absence (64nM) and presence (116nM) of saturating concentration of maltose. Moreover, the yield of soluble MBP-ATD2B is 18 times higher than the refolded 6xHis-ATD2B. We have reported a systematic comparison of three different affinity tagging strategies and identified a rapid and efficient method to obtain large amount of ATD2B recombinant protein for biochemical and structural studies.