Development of a selective competitive receptor binding assay for the determination of the affinity to NR2B containing NMDA receptors
Development of a selective competitive receptor binding assay for the determination of the affinity to NR2B containing NMDA receptors
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DOI：
10.1016/j.jpba.2010.04.014
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发表时间：
2010-11-02
影响因子：
3.4
通讯作者：
Wuensch, Bernhard
中科院分区：
文献类型：
--
作者：
Schepmann, Dirk;Frehland, Bastian;Wuensch, Bernhard
在该检测系统中，[H-3]伊芬普罗地尔被用作放射配体，而稳定表达重组人NR1a/NR2B受体的L（tk-）细胞的膜均浆作为受体材料，在检测过程中对细胞进行超声检测。通过饱和和结合/解离实验研究了结合动力学[H-3]，伊芬普罗地尔与人重组NR 1a/NR2B受体上的单个饱和位点结合。分离速率常数k（off）为0 03861 min(-1)，结合速率常数k（on）为0 00625 nM(-1) min（-1）。在未加地塞米松诱导细胞合成NMDA受体的细胞中证实了该方法的特异性。此外，α (1), sigma(1)和sigma(2)受体缺失。新方法测定的NR2B配体伊芬普罗地尔和埃利普罗地尔的k - i值与报道的数据吻合良好。(C) 2010 Elsevier b.v.版权所有
A selective, rapid and efficient competitive binding assay for the determination of the affinity of compounds towards the ifenprodil binding site of NR2B subunit containing NMDA receptors has been developed In the assay system, [H-3]ifenprodil was used as radioligand and membrane homogenates from L(tk-) cells stably expressing recombinant human NR1a/NR2B receptors served as the receptor material Sonication of the cells during work-up, performing all steps with 96-well multiplates and using a solid scintillator represent particular features of this assay The binding kinetics was investigated by saturation and association/dissociation experiments [H-3] ifenprodil bound to a single, saturable site on human recombinant NR 1a/NR2B receptors, resulting in a B-max-value of 258 pmol/mu g protein and K-d-value of 7.6 +/- 2 3 nM (SEM) The dissociation rate constant (k(off)) was 0 03861 min(-1) and the association rate constant k(on) resulted in 0 00625 nM(-1) min(-1) The specificity of the assay was proved with cells not treated with dexamethasone, which has to be added to induce NMDA receptor synthesis of the cells. Additionally, the absence of alpha(1), sigma(1) and sigma(2) receptors was shown. The K-I-values of the NR2B ligands ifenprodil and eliprodil determined with the new assay are in good accordance with reported data. (C) 2010 Elsevier B.V All rights reserved