Mapping the Ligand Binding Sites of Kainate Receptors: Molecular Determinants of Subunit-Selective Binding of the Antagonist [3H]UBP310
Mapping the Ligand Binding Sites of Kainate Receptors: Molecular Determinants of Subunit-Selective Binding of the Antagonist [3H]UBP310
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DOI：
10.1124/mol.110.067934
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发表时间：
2010-12-01
影响因子：
3.6
通讯作者：
Molnar, Elek
中科院分区：
文献类型：
--
作者：
Atlason, Palmi T.;Scholefield, Caroline L.;Molnar, Elek
红藻氨酸受体 (KAR) 调节突触传递和可塑性，其功能障碍与癫痫和慢性疼痛等多种疾病状态有关。 KAR 是由五个不同亚基形成的四聚体。 GluK1-3 是低亲和力红藻氨酸结合亚基，而 GluK4/5 以高亲和力结合红藻氨酸。许多这样的亚基可以存在于任何给定的细胞类型中，并且亚基的不同组合赋予KAR不同的特性。在这里，我们报告了使用人重组 KAR 的新型 GluK1 亚基选择性放射性标记拮抗剂 (S)-1-(2-氨基-2-羧乙基)-3-(2-羧基噻吩-3-基-甲基)-5-甲基嘧啶-2,4-二酮 ([H-3] UBP310) 的表征。 [H-3] UBP310 以低纳摩尔亲和力 (K-D = 21 +/- 7 nM) 与 GluK1 结合，但与 GluK2 没有特异性结合。然而，[H-3]UBP310 也与 GluK3 结合（K-D = 0.65 +/- 0.19 mu M），但亲和力比对 GluK1 观察到的亲和力低 30 倍。 GluK1 上的竞争 [H-3] UBP310 结合实验揭示了已知 GluK1 选择性配体的亲和力排序与先前在功能测定中报道的相同。在模型研究中被认为对于确定 UBP310 的 GluK1 选择性很重要的 GluK1-3 中的非保守残基进行点突变以在亚基之间切换残基。所有突变均未改变 KAR 亚基的表达或运输。 GluK1-T503A 突变减少了 [H-3]UBP310 结合，而 GluK2-A487T 突变则挽救了这种结合。同样，虽然 GluK1-N705S/S706N 突变减少，但 GluK3-N691S 突变增加了 [H-3]UBP310 结合活性。这些数据表明，GluK2 中的 Ala487 和 GluK3 中的 Asn691 是降低 UBP310 对这些亚基的亲和力的重要决定因素。这些建模和点突变研究的见解将有助于开发新的亚基选择性 KAR 拮抗剂。
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 ([H-3] UBP310) using human recombinant KARs. [H-3] UBP310 binds to GluK1 with low nanomolar affinity (K-D = 21 +/- 7 nM) but shows no specific binding to GluK2. However, [H-3]UBP310 also binds to GluK3 (K-D = 0.65 +/- 0.19 mu M) but with similar to 30-fold lower affinity than that observed for GluK1. Competition [H-3] 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 [H-3] UBP310 binding, GluK2-A487T mutation rescued it. Likewise, whereas GluK1-N705S/S706N mutation decreased, GluK3-N691S mutation increased [H-3] 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.