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Research ArticleArticle

Mapping the Ligand Binding Sites of Kainate Receptors: Molecular Determinants of Subunit-Selective Binding of the Antagonist [3H]UBP310

Palmi T. Atlason, Caroline L. Scholefield, Richard J. Eaves, M. Belen Mayo-Martin, David E. Jane and Elek Molnár
Molecular Pharmacology December 2010, 78 (6) 1036-1045; DOI: https://doi.org/10.1124/mol.110.067934
Palmi T. Atlason
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Caroline L. Scholefield
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Richard J. Eaves
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M. Belen Mayo-Martin
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David E. Jane
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Elek Molnár
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Abstract

Kainate receptors (KARs) modulate synaptic transmission and plasticity, and their dysfunction has been linked to several disease states such as epilepsy and chronic pain. KARs are tetramers formed from five different subunits. GluK1–3 are low affinity kainate binding subunits, whereas GluK4/5 bind kainate with high affinity. A number of these subunits can be present in any given cell type, and different combinations of subunits confer different properties to KARs. Here we report the characterization of a new GluK1 subunit-selective radiolabeled antagonist (S)-1-(2-amino-2-carboxyethyl)-3-(2-carboxythiophene-3-yl-methyl)-5-methylpyrimidine-2,4-dione ([3H]UBP310) using human recombinant KARs. [3H]UBP310 binds to GluK1 with low nanomolar affinity (KD = 21 ± 7 nM) but shows no specific binding to GluK2. However, [3H]UBP310 also binds to GluK3 (KD = 0.65 ± 0.19 μM) but with ∼30-fold lower affinity than that observed for GluK1. Competition [3H]UBP310 binding experiments on GluK1 revealed the same rank order of affinity of known GluK1-selective ligands as reported previously in functional assays. Nonconserved residues in GluK1–3 adjudged in modeling studies to be important in determining the GluK1 selectivity of UBP310 were point-mutated to switch residues between subunits. None of the mutations altered the expression or trafficking of KAR subunits. Whereas GluK1-T503A mutation diminished [3H]UBP310 binding, GluK2-A487T mutation rescued it. Likewise, whereas GluK1-N705S/S706N mutation decreased, GluK3-N691S mutation increased [3H]UBP310 binding activity. These data show that Ala487 in GluK2 and Asn691 in GluK3 are important determinants in reducing the affinity of UBP310 for these subunits. Insights from these modeling and point mutation studies will aid the development of new subunit-selective KAR antagonists.

Footnotes

  • This work was supported by the UK Medical Research Council [Grants G0601509, G0601812]; and the Biotechnology and Biological Sciences Research Council [Grant BB/F012519/1]. C.L.S. is a UK Medical Research Council-funded PhD student.

  • Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.

    doi:10.1124/mol.110.067934.

  • ABBREVIATIONS:

    KAR
    kainate receptor
    ACET
    (S)-1-(2-amino-2-carboxyethyl)-3-(2-carboxy-5-phenylthiophene-3-yl-methyl)-5-methylpyrimidine-2,4-dione
    AMPA
    (S)-2-amino-3-hydroxy-5-methyl-4-isoxazolepropanoic acid
    EndoF
    endoglycasidase F
    EndoH
    endoglycasidase H
    HEK
    human embryonic kidney
    KA
    kainate, (2S,3S,4S)-3-carboxymethyl-4-isopropenyl-pyrrolidine-2-carboxylic acid
    LBD
    ligand binding domain
    NMDA
    N-methyl-d-aspartate
    WT
    wild type
    ER
    endoplasmic reticulum
    PEI
    polyethylenimine
    NBQX
    2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(f)quinoxaline
    UBP310
    (S)-1-(2-amino-2-carboxyethyl)-3-(2-carboxythiophene-3-yl-methyl)-5-methylpyrimidine-2,4-dione
    UBP315
    (S)-1-(2-amino-2-carboxyethyl)-3-(2-carboxy-4,5-dibromothiophene-3-yl-methyl)-5-methylpyrimidine-2,4-dione
    UBP302
    (S)-1-(2-amino-2-carboxyethyl)-3-(2-carboxybenzyl)pyrimidine-2,4-dione
    UBP296
    (R,S)-1-(2-amino-2-carboxyethyl)-3-(2-carboxybenzyl)pyrimidine-2,4-dione
    UBP304
    (S)-1-(2-amino-2-carboxyethyl)-3-(2-carboxythiophene-3-ylmethyl)pyrimidine-2,4-dione).

  • Received July 30, 2010.
  • Accepted September 13, 2010.
  • Copyright © 2010 The American Society for Pharmacology and Experimental Therapeutics
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Molecular Pharmacology: 78 (6)
Molecular Pharmacology
Vol. 78, Issue 6
1 Dec 2010
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Research ArticleArticle

Mapping the Ligand Binding Sites of Kainate Receptors: Molecular Determinants of Subunit-Selective Binding of the Antagonist [3H]UBP310

Palmi T. Atlason, Caroline L. Scholefield, Richard J. Eaves, M. Belen Mayo-Martin, David E. Jane and Elek Molnár
Molecular Pharmacology December 1, 2010, 78 (6) 1036-1045; DOI: https://doi.org/10.1124/mol.110.067934

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Research ArticleArticle

Mapping the Ligand Binding Sites of Kainate Receptors: Molecular Determinants of Subunit-Selective Binding of the Antagonist [3H]UBP310

Palmi T. Atlason, Caroline L. Scholefield, Richard J. Eaves, M. Belen Mayo-Martin, David E. Jane and Elek Molnár
Molecular Pharmacology December 1, 2010, 78 (6) 1036-1045; DOI: https://doi.org/10.1124/mol.110.067934
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