GABAA receptor open-state conformation determines non-competitive antagonist binding.
GABAA receptor open-state conformation determines non-competitive antagonist binding.
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GABAA 受体开放态构象决定非竞争性拮抗剂结合。
DOI：
10.1016/j.taap.2010.11.010
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发表时间：
2011
影响因子：
3.8
通讯作者：
Casida,JohnE
中科院分区：
文献类型：
--
作者：
Chen,Ligong;Xue,Ling;Giacomini,KathleenM;Casida,JohnE
γ-氨基丁酸A型受体是最重要的杀虫靶标之一。人重组β-3均聚体是该结合位点的最佳模型，4-N-[~H]丙基-4‘-乙炔基双环苯甲酸([~H]EBOB)是首选的非竞争性拮抗剂放射性配体。β3均聚体相对于活性低得多但结构上非常相似的β1均聚体具有独特的高敏感性，为阐明对NCA结合重要的结构和功能特征提供了理想的比较。通过嵌合和突变以及与β1亚基和调节剂的不同组合对β1和α3亚基进行比较。嵌合体β3/β1具有β3亚单位胞外区和β1亚基跨膜螺旋，保持了β3同聚体的高结合水平，而嵌合体β1/β3与β1亚单位胞外区和β3亚单位跨膜螺旋具有与β1相同的低结合活性。3μM的GABA通过增加通道的开放概率，刺激异构体α1β1和α1β3的结合水平2倍以上。α1亚基的加入挽救了失活的β1/β3嵌合体，接近野生型α1β1的活性。突变β1S15‘N和β3N15’S分别与野生型β1和β3相比显著改变了eBOB的结合。但是，α1β1S15‘N的结合活性对GABA不敏感，而α1β3N15’S被GABA刺激的程度远低于野生型α1β3。依托咪酯对NCA结合的抑制作用被突变β3N15‘s降低了5倍以上。因此，NCA结合部位受到开放状态构象的严格调控，这在很大程度上决定了GABA受体的敏感性。
The γ-aminobutyric acid (GABA) type A receptor (GABAAR) is one of the most important targets for insecticide action. The human recombinant β3 homomer is the best available model for this binding site and 4-n-[3H]propyl-4′-ethynylbicycloorthobenzoate ([3H]EBOB) is the preferred non-competitive antagonist (NCA) radioligand. The uniquely high sensitivity of the β3 homomer relative to the much-less-active but structurally very-similar β1 homomer provides an ideal comparison to elucidate structural and functional features important for NCA binding. The β1 and β3 subunits were compared using chimeragenesis and mutagenesis and various combinations with the α1 subunit and modulators. Chimera β3/β1 with the β3 subunit extracellular domain and the β1 subunit transmembrane helices retained the high [3H]EBOB binding level of the β3 homomer while chimera β1/β3 with the β1 subunit extracellular domain and the β3 subunit transmembrane helices had low binding activity similar to the β1 homomer. GABA at 3μM stimulated heteromers α1β1 and α1β3 binding levels more than 2-fold by increasing the open probability of the channel. Addition of the α1 subunit rescued the inactive β1/β3 chimera close to wildtype α1β1 activity. EBOB binding was significantly altered by mutations β1S15′N and β3N15′S compared with wildtype β1 and β3, respectively. However, the binding activity of α1β1S15′N was insensitive to GABA and α1β3N15′S was stimulated much less than wildtype α1β3 by GABA. The inhibitory effect of etomidate on NCA binding was reduced more than 5-fold by the mutation β3N15′S. Therefore, the NCA binding site is tightly regulated by the open-state conformation that largely determines GABAAreceptor sensitivity.
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