The NR 1 M 3 Domain Mediates Allosteric Coupling in the N-Methyl-D-aspartate Receptor

Content Provider	Semantic Scholar
Author	Blanke, Marie Dongen, Antonius M. J. Van
Copyright Year	2008
Abstract	N-Methyl-D-aspartate (NMDA) receptors play a critical role in both development of the central nervous system and adult neuroplasticity. However, although the NMDA receptor presents a valuable therapeutic target, the relationship between its structure and functional properties has yet to be fully elucidated. To further explore the mechanism of receptor activation, we characterized two gain-of-function mutations within the NR1 M3 segment, a transmembrane domain proposed to couple ligand binding and channel opening. Both mutants (A7Q and A7Y) displayed significant glycine-independent currents, indicating that their M3 domains may preferentially adopt a more activated conformation. Substituted cysteine modification experiments revealed that the glycine binding clefts of both A7Q and A7Y are inaccessible to modifying reagents and resistant to competitive antagonism. These data suggest that perturbation of M3 can stabilize the ligand binding domain in a closed cleft conformation, even in the absence of agonist. Both mutants also displayed significant glutamate-independent current and insensitivity to glutamate-site antagonism, indicating partial activation by either glycine or glutamate alone. Furthermore, A7Q and A7Y increased accessibility of the NR2 M3 domain, providing evidence for intersubunit coupling at the transmembrane level and suggesting that these NR1 mutations dominate the properties of the intact heteromeric receptor. The equivalent mutations in NR2 did not exhibit comparable phenotypes, indicating that the NR1 and NR2 M3 domains may play different functional roles. In summary, our data demonstrate that the NR1 M3 segment is functionally coupled to key structural domains in both the NR1 and NR2 subunits. N-methyl-D-aspartate (NMDA) receptors are a critical component of the glutamatergic signaling system, responsible for generating excitatory synaptic currents that control neural transmission (Collingridge and Bliss, 1995). Long-term changes in synaptic strength, believed to underlie learning and memory formation, are induced by NMDA receptor-mediated calcium influx (Malenka and Nicoll, 1999). Consequently, receptor hypofunction has been implicated in many neurological disorders, ranging from cognition defects to schizophrenia and Alzheimer’s disease (Weeber and Sweatt, 2002; Emamian et al., 2004; Hynd et al., 2004). Overstimulation of NMDA receptors, which can result from excessive glutamate release after stroke or traumatic brain injury, triggers excitotoxic cell death (Mody and MacDonald, 1995), thus illustrating the importance of precise regulation. NMDA receptor activation requires concurrent glutamate release and membrane depolarization, as well as binding of a coagonist, glycine (Mayer et al., 1984; Kleckner and Dingledine, 1988). In addition, NMDA receptors are regulated by a variety of endogenous modulators, including protons, Zn , polyamines, and neurosteroids (Dingledine et al., 1999; Stoll et al., 2007). At the clinical level, pharmacological inhibition of glutamate receptors has been used for anesthesia and treatment of stroke and Alzheimer’s disease (Muir, 2006). Given the broad therapeutic potential of NMDA receptor antagonists and their diverse pharmacology, more detailed structure-function studies should prove extremely valuable. NMDA receptors generally form as heteromultimers of NR1 and NR2 subunits, which bind glycine and glutamate, respectively (Schorge and Colquhoun, 2003). Each subunit has the same modular domain structure, including a ligand binding domain (LBD), three putative transmembrane segments (M1, M3, and M4), and a pore-forming M2 loop (Fig. 1) (Mayer, 2006). Recent crystal structures of the LBDs have helped elucidate ligand binding and cleft closure (Furukawa This work was supported by National Institute of Health grants F31NS053030-01 (to M.L.B.) and R01-MH61506 (to A.M.J.V.D.). Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.107.044115. ABBREVIATIONS: NMDA, N-methyl-D-aspartate; LBD, ligand binding domain; GluR, glutamate receptor; LC-MS, liquid chromatography-mass spectrometry; Lobar, low-barium Ringer’s solution; MTSEA, methanethiosulfonate; DCK, 5,7-dichlorokynurenic acid; PS, pregnenolone sulfate; DCS, D-cycloserine; HA-966, 3-amino-1-hydroxy-2-pyrrolidinone; WT, wild type; DTNB, dithionitrobenzoic acid; APV, 2-amino-5-phosphonovalerate; MK-801, 5H-dibenzo[a,d]cyclohepten-5,10-imine (dizocilpine maleate); Po, open probability. 0026-895X/08/7402-454–465$20.00 MOLECULAR PHARMACOLOGY Vol. 74, No. 2 Copyright © 2008 The American Society for Pharmacology and Experimental Therapeutics 44115/3364984 Mol Pharmacol 74:454–465, 2008 Printed in U.S.A. 454 by gest on M arch 7, 2012 m oharm .aspeurnals.org D ow nladed fom 4115.DC1.html http://molpharm.aspetjournals.org/content/suppl/2008/12/04/mol.107.04 Supplemental Material can be found at: http://molpharm.aspetjournals.org/content/75/1/254.full.pdf An erratum has been published: et al., 2005), whereas single-channel studies have provided a detailed analysis of gating behavior (Banke and Traynelis, 2003; Auerbach and Zhou, 2005); however, structural information on the transmembrane region remains sparse, and the mechanism linking binding of agonists, antagonists, and modulators to channel gating is not fully understood. Previous studies have suggested that the M3 segment functions as a transduction element, coupling ligand binding to channel opening (Jones et al., 2002; Yuan et al., 2005). M3 contains the highly conserved SYTANLAAF motif, and many cysteine mutations in this region exhibit state-dependent accessibility, implying that M3 movement occurs in response to activation. Recent studies have also shown that M3 is the only transmembrane domain contributing to the deepest portion of the pore, supporting a prominent role in gating (Sobolevsky et al., 2007). In vivo, the lurcher mouse phenotype, a neurodegenerative condition caused by excitotoxic cell death, has been attributed to a constitutive mutation within the GluR 2 SYTANLAAF region (A8T) (Zuo et al., 1997), and introduction of the lurcher mutation into other glutamate receptors results in numerous gain-of-function phenotypes, including increased agonist potency, slower deactivation rates, and inhibition of desensitization (Kohda et al., 2000; Klein and Howe, 2004; Hu and Zheng, 2005; Schmid et al.,
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