Prior work employing functional analysis, photolabeling, and X-ray crystallography have identified three distinct binding sites for potentiating steroids in the heteromeric GABA_A receptor. The sites are located in the membrane-spanning domains of the receptor at the β-α subunit interface (site I) and within the α (site II) and β subunits (site III). Here, we have investigated the effects of mutations to these sites on potentiation of the rat α1β2γ2L GABA_A receptor by the endogenous neurosteroid allopregnanolone (3α5αP). The mutations were introduced alone or in combination to probe the additivity of effects. We show that the effects of amino acid substitutions in sites I and II are energetically additive, indicating independence of the actions of the two steroid binding sites. In site III, none of the mutations tested reduced potentiation by 3α5αP, nor did a mutation in site III modify the effects of mutations in sites I or II. We infer that the binding sites for 3α5αP act independently. The independence of steroid action at each site is supported by photolabeling data showing that mutations in either site I or site II selectively change steroid orientation in the mutated site without affecting labeling at the unmutated site. The findings are discussed in the context of linking energetic additivity to empirical changes in receptor function and ligand binding.

中文翻译：

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亚基内和亚基间类固醇结合位点独立地和加性地介导内源性神经类固醇异孕酮对α1β2γ2L GABAA受体的增强作用

先前采用功能分析、光标记和 X 射线晶体学的工作已经确定了三个不同的结合位点，用于增强异聚 GABA _A受体中的类固醇。这些位点位于受体的跨膜结构域中的β - α亚基界面（位点 I）和α（位点 II）和β亚基（位点 III）内。在这里，我们研究了这些位点的突变对大鼠α 1 β 2 γ 2L GABA _A受体通过内源性神经甾体异孕酮 (3 α 5 αP)。单独或组合引入突变以探测效应的可加性。我们表明位点 I 和 II 中氨基酸取代的影响是能量相加的，表明两个类固醇结合位点的作用是独立的。在位点 III 中，所测试的突变均未降低 3 α 5 α P 的增强作用，位点 III 中的突变也没有改变位点 I 或 II 中突变的影响。我们推断 3 α 5 α的结合位点P 独立行动。每个位点类固醇作用的独立性得到了光标记数据的支持，这些数据表明位点 I 或位点 II 的突变选择性地改变了突变位点的类固醇方向，而不影响未突变位点的标记。在将能量可加性与受体功能和配体结合的经验变化联系起来的背景下讨论了这些发现。