Identification of Amino Acid Residues of the NR2A Subunit That Control Glutamate Potency in Recombinant NR1/NR2A NMDA Receptors

Anson, Lesley; Chen, Philip E.; Wyllie, DJ; Colquhoun, David; Schoepfer, Ralf

doi:10.1523/jneurosci.18-02-00581.1998

Cited by 179 publications

(183 citation statements)

References 32 publications

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“…Serine to alanine mutations at this position in AMPA and NMDA receptors reportedly has more modest effects on glutamate affinity, and A689S mutation in GluR6 has no affect on glutamate mediated desensitization or 3 H-kainate binding. 22,23,32 In contrast to previous studies examining the reverse mutations in other iGluRs, we found no effect of A518T mutation in GluR6 on glutamate affinity or response kinetics. Neither did the A689S mutation affect these measures.…”

Section: Discussioncontrasting

confidence: 99%

“…5 Likewise for NMDA receptors, NR2B-S664G mutation (equivalent to GluR6-A689) reduced glutamate potency by 100-fold relative to wild-type 22 and NR2A-T671A (equivalent to GluR6-T690) reduced glutamate potency by 1000-fold. 23 It is striking, despite the natural substitutions of secondary ligand-binding residues, that AMPA and KARs have similar affinities and exhibit similar, rapidly desensitizing responses to their endogenous agonist, glutamate. Nonetheless, they exhibit significant functional and pharmacological differences.…”

Section: Introductionmentioning

confidence: 99%

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Functional Consequences of Natural Substitutions in the GluR6 Kainate Receptor Subunit Ligand-Binding Site

Kistler¹,

Fleck²

2007

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Differences in binding-site residues of GluR2 (AMPAR) and GluR6 (KAR) subunits have been identified that might account for their functional and pharmacological differences. Specifically, residues A518, A689 and N721 in GluR6 replace highly conserved threonine and serine residues found in other ionotropic glutamate receptor (iGluR) subunits. To define how these natural substitutions impact GluR6 function, we used patch clamp recording with ultrafast perfusion to characterize the effects of A518T, A689S and N721T on agonist potency, efficacy and response kinetics. We find these natural substitutions impact GluR6 function less than would be expected from reverse mutations in other iGluRs. There was little effect of individual or combined mutations on glutamate potency, deactivation or desensitization kinetics. Altered recovery kinetics were seen that were greatest after combined mutations. Kainate potency and response kinetics were also unchanged in the mutants, whereas kainate efficacy was reduced in A518T and increased the T/S/T mutant relative glutamate. Notably, A518T and A689S mutation permitted AMPA to bind as a weak competitive antagonist and the effects of these mutations were additive. N721T mutation further enhanced AMPA binding, allowing AMPA to activate and fully desensitize the receptors. Alternative mutations altering side chain length at position 518 produced far greater changes in glutamate affinity and response kinetics than did the natural mutations. We conclude that these nonconserved residues in GluR6 define the size of the agonist-binding pocket, exerting a steric influence on the bound agonist and the extent of binding-domain closure that can influence agonist potency, deactivation, desensitization and recovery kinetics.

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Section: Discussioncontrasting

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Functional Consequences of Natural Substitutions in the GluR6 Kainate Receptor Subunit Ligand-Binding Site

Kistler¹,

Fleck²

2007

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“…The residue homologous to GluR2-R485 is perfectly conserved in all AMPA, KA, and NMDA subunits and may be the principal residue to bind during the initial docking step of agonist association (Abele et al, 2000;Armstrong and Gouaux, 2000). Mutagenesis studies confirm the equivalent residue is critical for agonist-induced function in AMPA (Uchino et al, 1992), KA (Ayalon and Stern-Bach, 2001), and NMDA (Laube et al, 1997;Anson et al, 1998) receptors. The residue homologous to GluR2-T655 is also conserved in AMPA, KA, and NMDA subunits, except KA1, which substitutes a serine, and NR1, which substitutes a valine at this position and confers its altered selectivity for glycine (Furukawa and Gouaux, 2003).…”

Section: Glur6-binding Residuesmentioning

confidence: 94%

“…The residue homologous to GluR2-T655 is also conserved in AMPA, KA, and NMDA subunits, except KA1, which substitutes a serine, and NR1, which substitutes a valine at this position and confers its altered selectivity for glycine (Furukawa and Gouaux, 2003). The T655 interaction with agonist is underscored by alanine mutagenesis studies of both AMPA and NR2 receptors (Anson et al, 1998;Lampinen et al, 2002). Likewise, the residue homologous to GluR2-E705 is perfectly conserved in all AMPA and KA subunits and is critical to agonist-receptor interactions in AMPA receptors (Mano et al, 1996;Lampinen et al, 1998;Abele et al, 2000).…”

Section: Glur6-binding Residuesmentioning

confidence: 99%

Glutamate Receptor Trafficking: Endoplasmic Reticulum Quality Control Involves Ligand Binding and Receptor Function

Mah

Cornell²,

Mitchell³

et al. 2005

J. Neurosci.

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The glutamate receptor (GluR) agonist-binding site consists of amino acid residues in the extracellular S1 and S2 domains in the N-terminal and M3-M4 loop regions, respectively. In the present study, we sought to confirm that the conserved ligand-binding residues identified in the AMPA receptor S1S2 domains also participate in ligand binding of GluR6 kainate receptors. Amino acid substitutions were made in the GluR6 parent at R523, T690, and E738 to alter their potential interactions with ligand. Mutant receptors were expressed in human embryonic kidney 293 cells, confirmed by Western blot analysis, and tested by [

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“…In contrast, the mutation T677A strongly affected the binding of all three agonists. The side chain of this threonine has direct hydrogen bonding interactions with the agonists, and noticeable effects on agonist affinities by mutations at the corresponding residue have been reported previously (6,23).…”

Section: Resultsmentioning

confidence: 65%

Discrimination between Agonists and Antagonists by the α-Amino-3-hydroxy-5-methyl-4-isoxazole Propionic Acid-selective Glutamate Receptor

Lampinen¹,

Settimo²,

Pentikäinen³

et al. 2002

Journal of Biological Chemistry

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The ligand-binding site of ionotropic glutamate receptors is composed of two extracellular segments, S1 and S2, homologous to bacterial amino acid-binding proteins (1-3). Recent determination of the crystal structure of an S1S2 fusion protein of the GluR-B 1 (GluR2) ␣-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor subunit as complexed with ligands (4, 5) provided the first atomic resolution view into a neurotransmitter-binding pocket and confirmed the earlier predictions of a close structural and functional similarity to the bacterial proteins (1-3, 6 -9). The agonists kainate, glutamate, and AMPA are engaged in multiple polar and van der Waals contacts that stabilize a closed state of the two-lobed binding domain (4, 5). The charged ␣-aminocarboxylate group of glutamate and AMPA and the pyrrolidine carboxyl and imino groups of kainate are accommodated by hydrogen bonds and ion pair interactions with the oppositely charged side chains of Arg-485 and Glu-705, respectively. The distal negatively charged group of the agonists, the carboxylmethyl group of kainate, the ␥-carboxylate of glutamate, or the isoxazole ring hydroxyl of AMPA, make close polar contacts with the base of an ␣-helix ("helix F") in the lobe 2, but the exact hydrogen bonding patterns differ between agonists (4,5). In contrast to the agonist complexes, the ligand-free apo form and one antagonist complex (6,7-dinitroquinoxaline-2,3-dione (DNQX)) of GluR-B S1S2 assume a more open state, suggesting that agonist activity is based on their ability to induce a slight closure of the lobes (5).Although the stereochemical features of the agonist-receptor interaction revealed by the crystal structure are largely supported by mutagenesis data, not much is yet known on how the receptor discriminates between agonists and antagonists or on how structurally different antagonists interact with the receptor. In particular, it is currently unclear to what extent the contacts antagonists make with the receptor overlap those made by agonists. Although previous mutagenesis work has identified amino acid residues that affect antagonist affinities in the AMPA receptor, for example , no mutations that would selectively affect only agonists or antagonists have been reported. As a step toward understanding the structural basis of discrimination between agonists and antagonists by AMPA receptors, we have analyzed the ligand binding properties of the mutated GluR-D ligand-binding domain constructs by using [ 3 H]AMPA, an agonist, and [ 3 H]Ro 48-8587, a high affinity AMPA receptor an-* This study was financially supported by the Academy of Finland and the National Technology Agency of Finland (TEKES). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.ʈ To whom correspondence should be addressed: Dept. of Biosciences, Viikinkaari 5D, 00014 University of Helsinki, Helsinki, Finland....

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Identification of Amino Acid Residues of the NR2A Subunit That Control Glutamate Potency in Recombinant NR1/NR2A NMDA Receptors

Cited by 179 publications

References 32 publications

Functional Consequences of Natural Substitutions in the GluR6 Kainate Receptor Subunit Ligand-Binding Site

Functional Consequences of Natural Substitutions in the GluR6 Kainate Receptor Subunit Ligand-Binding Site

Glutamate Receptor Trafficking: Endoplasmic Reticulum Quality Control Involves Ligand Binding and Receptor Function

Discrimination between Agonists and Antagonists by the α-Amino-3-hydroxy-5-methyl-4-isoxazole Propionic Acid-selective Glutamate Receptor

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