Mutation of the GABAA receptor M1 transmembrane proline increases GABA affinity and reduces barbiturate enhancement
Mutation of the GABAA receptor M1 transmembrane proline increases GABA affinity and reduces barbiturate enhancement
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DOI：
10.1016/s0028-3908(01)00196-4
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发表时间：
2002-03-01
影响因子：
4.7
通讯作者：
Macdonald, RL
中科院分区：
文献类型：
--
作者：
Greenfield, LJ;Zaman, SH;Macdonald, RL
所有GABA（A）受体（GABAR）亚单位在第一跨膜区段（MI）的共有基序中包括不变的脯氨酸。在含有牛α 1、β 1和γ 2亚基的受体中，我们使用3种不同的表达系统分析了在α 1或β 1亚基中将该M I脯氨酸突变为丙氨酸的效果。在HEK 293细胞和L929成纤维细胞的全细胞记录中，β 1亚基突变体β 1（P228 A）使GABA的EC 50降低约10倍。当在爪蟾卵母细胞中表达时，相应的α 1亚基突变体（α 1（P233 A））也降低了GABA EC 50：α 1（P233 A）β 1 γ 2S受体在HEK 293细胞中未能组装。[H-3]氟马西尼和[H-3]蝇蕈醇与转染的HEK 293细胞膜的结合显示，与含有β 1或β 1（P228 A）亚基的GABAR的受体表达水平相似，但对[H-3]氟马西尼的亲和力没有变化。在含有β 1（P228 A）亚基的GABAR中，对[H-3]蝇蕈醇的亲和力增加了6倍。在L929细胞中，β 1（P228 A）亚基的存在降低了巴比妥类药物的增强作用，而不影响地西泮或阿法沙龙的增强作用。单通道记录alpha 1 beta1 gamma 2S和alpha 1 beta1（P228 A）gamma 2L GABARs显示类似的通道动力学。但含有β-突变体的受体在较低的GABA浓度下打开。我们的结论是β 1亚基MI段脯氨酸影响GABA结合和通道门控之间的联系，是巴比妥类药物增强的关键。α 1亚基中M 1脯氨酸的突变也抑制受体组装。(C)2002爱思唯尔科技有限公司版权所有。
All GABA(A) receptor (GABAR) Subunits include an invariant proline in a consensus motif in the first transmembrane segment (MI). In receptors containing bovine alpha1, beta1 and gamma2 Subunits, we analyzed the effect of mutating this M I proline to alanine in the alpha1 or beta1 Subunit using 3 different expression systems. The beta1 Subunit mutant, beta1(P228A), reduced the EC50 for GABA about 10-fold in whole cell recordings in HEK293 cells and L929 fibroblasts. The corresponding alpha1 subunit mutant (alpha1(P233A)) also reduced the GABA EC50 when expressed in Xenopus oocytes: alpha1(P233A)beta1gamma2S receptors failed to assemble in HEK293 cells. Binding of [H-3]flumazenil and [H-3]muscimol to transfected HEK293 cell membranes showed similar levels of receptor expression with GABARs containing beta1 or beta1(P228A) subunits and no change in the affinity for [H-3]flumazenil: however. the affinity for [H-3]muscimol was increased 6-fold in GABARs containing beta1(P228A) Subunits. In L929 cells, presence of the beta1(P228A) Subunit reduced enhancement by barbiturates Without affecting enhancement by diazepam or alfaxalone. Single channel recordings front alpha1beta1gamma2S and alpha1beta1(P228A)gamma2L GABARs showed similar channel kinetics. but beta-mutant containing receptors opened at lower GABA concentrations. We Conclude that the beta1 Subunit MI segment proline affects the linkage between GABA binding and channel gating and is critical for barbiturate enhancement. Mutation of the M I proline in the alpha1 Subunit also inhibited receptor assembly. (C) 2002 Elsevier Science Ltd. All rights reserved.