Functional characterization of neurotransmitter activation and modulation in a nematode model ligand-gated ion channel.
Functional characterization of neurotransmitter activation and modulation in a nematode model ligand-gated ion channel.
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DOI：
10.1111/jnc.13644
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发表时间：
2016-07
影响因子：
4.7
通讯作者：
Howard RJ
中科院分区：
文献类型：
--
作者：
Heusser SA;Yoluk Ö;Klement G;Riederer EA;Lindahl E;Howard RJ
五聚体配体门控离子通道的超家族包括在脊椎动物中介导快速突触传递的神经递质受体，并且是包括酒精、麻醉剂、苯二氮卓类和抗惊厥剂的药物的靶标。然而，尽管这些受体具有药理学重要性，但其离子通道开放、门控和调节机制仍存在许多悬而未决的问题。细胞外和跨膜结构域的细微构象变化可能会影响通道开放，但由于人类膜蛋白的结构数据有限，因此难以表征。最近修饰的秀丽隐杆线虫谷氨酸门控氯离子通道（GluCl）在多个状态下的晶体结构为结构-功能研究提供了一个有吸引力的模型系统。然而，晶体学GluCl构建体的药理学尚未完全确立。为了建立该系统的功能相关性，我们使用了两个电极电压钳电生理学在非洲爪蟾卵母细胞的晶体学和本地样GluCl结构的L-谷氨酸盐和伊维菌素的特征激活。我们还测试了乙醇和其他麻醉剂的调制，并使用定点诱变来探索环F的一个区域的作用，该区域通过分子动力学模拟参与配体门控。我们的研究结果表明，晶体结构功能模型的浓度依赖性激动和变构调节的ESTA相关受体。在环F中的残基Leu 174处的特定取代改变了直接的L-谷氨酸激活，这与该区域在配体结合中的作用的计算证据一致。这些见解表明，在这个受体家族的激活和调制特性的保护，并建立一个框架，GluCl作为一个模型系统，包括新的药物发现的可能性。在这项研究中，我们阐明了修饰的谷氨酸门控氯离子通道（GluClcryst）作为GABAA受体结构上可及模型的有效性。与天然样对照相比，GluClcryst通过其神经递质配体L-谷氨酸表现出经典的激活作用。修饰的通道也对与人GABAA受体相关的变构调节剂敏感，并且对预测改变通道开放的定点突变敏感。
The superfamily of pentameric ligand-gated ion channels includes neurotransmitter receptors that mediate fast synaptic transmission in vertebrates, and are targets for drugs including alcohols, anesthetics, benzodiazepines and anticonvulsants. However, the mechanisms of ion channel opening, gating and modulation in these receptors leave many open questions, despite their pharmacological importance. Subtle conformational changes in both the extracellular and transmembrane domains are likely to influence channel opening, but have been difficult to characterize given the limited structural data available for human membrane proteins. Recent crystal structures of a modified Caenorhabditis elegans glutamate-gated chloride channel (GluCl) in multiple states offer an appealing model system for structure-function studies. However, the pharmacology of the crystallographic GluCl construct is not well established. To establish the functional relevance of this system, we used two-electrode voltage-clamp electrophysiology in Xenopus oocytes to characterize activation of crystallographic and native-like GluCl constructs by L-glutamate and ivermectin. We also tested modulation by ethanol and other anesthetic agents, and used site-directed mutagenesis to explore the role of a region of Loop F which was implicated in ligand gating by molecular dynamics simulations. Our findings indicate that the crystallographic construct functionally models concentration-dependent agonism and allosteric modulation of pharmacologically relevant receptors. Specific substitutions at residue Leu174 in loop F altered direct L-glutamate activation, consistent with computational evidence for this region’s role in ligand binding. These insights demonstrate conservation of activation and modulation properties in this receptor family, and establish a framework for GluCl as a model system, including new possibilities for drug discovery. In this study, we elucidate the validity of a modified glutamate-gated chloride channel (GluClcryst) as a structurally accessible model for GABAA receptors. In contrast to native-like controls, GluClcryst exhibits classical activation by its neurotransmitter ligand L-glutamate. The modified channel is also sensitive to allosteric modulators associated with human GABAA receptors, and to site-directed mutations predicted to alter channel opening.