Laboratory of Neurophysiology, Department of Physiology (C.F.B., .P.A.C., T.M., L.G.A.), Laboratory of Molecular Neurobiology, Department of Physiology (L.G.), Laboratory of Molecular Biophysics, Department of Biochemistry and Molecular Biology (M.B.), and Ph.D. program in Pharmacology (T.M.), University of Concepción, Concepción, Chile.
Laboratory of Neurophysiology, Department of Physiology (C.F.B., .P.A.C., T.M., L.G.A.), Laboratory of Molecular Neurobiology, Department of Physiology (L.G.), Laboratory of Molecular Biophysics, Department of Biochemistry and Molecular Biology (M.B.), and Ph.D. program in Pharmacology (T.M.), University of Concepción, Concepción, Chile
J Pharmacol Exp Ther. 2015 Jan;352(1):148-55. doi: 10.1124/jpet.114.217976. Epub 2014 Oct 22.
The α1-subunit containing glycine receptors (GlyRs) is potentiated by ethanol, in part, by intracellular Gβγ actions. Previous studies have suggested that molecular requirements in the large intracellular domain are involved; however, the lack of structural data about this region has made it difficult to describe a detailed mechanism. Using circular dichroism and molecular modeling, we generated a full model of the α1-GlyR, which includes the large intracellular domain and provides new information on structural requirements for allosteric modulation by ethanol and Gβγ. The data strongly suggest the existence of an α-helical conformation in the regions near transmembrane (TM)-3 and TM4 of the large intracellular domain. The secondary structure in the N-terminal region of the large intracellular domain near TM3 appeared critical for ethanol action, and this was tested using the homologous domain of the γ2-subunit of the GABAA receptor predicted to have little helical conformation. This region of γ2 was able to bind Gβγ and form a functional channel when combined with α1-GlyR, but it was not sensitive to ethanol. Mutations in the N- and C-terminal regions introduced to replace corresponding amino acids of the α1-GlyR sequence restored the ability to be modulated by ethanol and Gβγ. Recovery of the sensitivity to ethanol was associated with the existence of a helical conformation similar to α1-GlyR, thus being an essential secondary structural requirement for GlyR modulation by ethanol and G protein.
α1 亚基含甘氨酸受体（GlyRs）可被乙醇增强，部分原因是细胞内 Gβγ 作用。先前的研究表明，该受体的大细胞内结构域涉及分子要求；然而，由于缺乏该区域的结构数据，很难描述详细的机制。我们使用圆二色性和分子建模生成了完整的α1-GlyR 模型，其中包括大细胞内结构域，并提供了关于乙醇和 Gβγ 变构调节的结构要求的新信息。数据强烈表明，大细胞内结构域的 TM-3 和 TM4 附近区域存在α-螺旋构象。大细胞内结构域 TM3 附近 N 末端区域的二级结构对于乙醇作用至关重要，这是使用 GABAA 受体 γ2 亚基的同源结构域进行测试的，该结构域被预测具有很少的螺旋构象。当与α1-GlyR 结合时，该 γ2 区域的该部分能够结合 Gβγ 并形成功能性通道，但它对乙醇不敏感。引入替换α1-GlyR 序列的 N 和 C 末端区域的突变恢复了被乙醇和 G 蛋白调节的能力。恢复对乙醇的敏感性与存在类似α1-GlyR 的螺旋构象有关，因此是乙醇和 G 蛋白调节 GlyR 的必需二级结构要求。
