Environmental Chemistry and Toxicology Laboratory, Department of Environmental Science, Policy and Management, University of California, Berkeley, CA 94720, USA.
Toxicol Appl Pharmacol. 2011 Feb 1;250(3):221-8. doi: 10.1016/j.taap.2010.11.010. Epub 2010 Nov 25.
The γ-aminobutyric acid (GABA) type A receptor (GABA(A)R) 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-[(3)H]propyl-4'-ethynylbicycloorthobenzoate ([(3)H]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 [(3)H]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 GABA(A) receptor sensitivity.
γ-氨基丁酸（GABA）A型受体（GABA(A)R）是杀虫剂作用的最重要靶标之一。人源重组β3 同源四聚体是该结合位点最可用的模型，4-n-[(3)H]丙基-4'-炔基双环邻苯二甲酸酯（[(3)H]EBOB）是首选的非竞争性拮抗剂（NCA）放射性配体。β3 同源四聚体相对于结构非常相似但活性低得多的β1 同源四聚体具有极高的灵敏度，为阐明对 NCA 结合很重要的结构和功能特征提供了理想的比较。使用嵌合和突变以及与α1 亚基和调节剂的各种组合比较了β1 和β3 亚基。β3 亚基胞外域和β1 亚基跨膜螺旋的嵌合体β3/β1 保留了β3 同源四聚体的高[(3)H]EBOB 结合水平，而β1 亚基胞外域和β3 亚基跨膜螺旋的嵌合体β1/β3 具有与β1 同源四聚体相似的低结合活性。GABA 在 3μM 时通过增加通道的开放概率，使异源二聚体α1β1 和α1β3 的结合水平增加了 2 倍以上。添加α1 亚基使无活性的β1/β3 嵌合体接近野生型α1β1 的活性。与野生型β1 和β3 相比，突变β1S15'N 和β3N15'S 显著改变了 EBOB 结合。然而，GABA 对α1β1S15'N 的结合活性没有影响，而 GABA 对α1β3N15'S 的刺激作用远小于野生型α1β3。突变β3N15'S 使乙咪酯对 NCA 结合的抑制作用降低了 5 倍以上。因此，NCA 结合位点受开放状态构象的严格调节，该构象在很大程度上决定了 GABA(A)受体的敏感性。
