Ethanol Modulation is Quantitatively Determined by the Transmembrane Domain of Human α1 Glycine Receptors.
Ethanol Modulation is Quantitatively Determined by the Transmembrane Domain of Human α1 Glycine Receptors.
复制标题
乙醇调节是由人类α1 甘氨酸受体的跨膜域定量确定的。
DOI：
10.1111/acer.12735
复制
发表时间：
2015
期刊：
影响因子：
--
通讯作者：
Howard,RebeccaJ
中科院分区：
文献类型：
--
作者：
Horani,Suzzane;Stater,EvanP;Corringer,Pierre-Jean;Trudell,JamesR;Harris,RAdron;Howard,RebeccaJ
背景突变和标记研究已经从人α1甘氨酸受体（GlyR）的细胞外、跨膜（TM）和细胞内结构域中鉴定出介导乙醇（EtOH）增强作用的氨基酸。然而，该Cys环受体超家族中生理学相关受体的有限高分辨率结构数据使得难以精确定位关键氨基酸。来自低等生物的Homoprotein离子通道为Cys环受体的结构和功能特性提供了保守的模型。我们之前证明了一个单一的氨基酸变异的Gloeavolaceus配体门控离子通道（GLIC），产生的EtOH和麻醉敏感性与GlyRs相似，并提供了EtOH与GLIC结合的晶体学证据。方法我们使用2电极电压钳电生理学直接比较了α1 GlyR和GLIC的EtOH调节与含有人α1 GlyRs的TM结构域和GLIC的配体结合结构域的嵌合体。结果乙醇在锌螯合剂存在下以浓度依赖性方式增强α1 GlyRs，但在高浓度下不增强GLIC。GLIC/GlyR嵌合体概括了GlyR的EtOH增强作用，对锌螯合没有明显的敏感性。对于嵌合体在卵母细胞中的表达，这是必不可少的，以抑制泄漏电流，通过添加50μM印防己毒素的媒体，一种技术，可能有应用程序在其他离子channels.ConclusionsOur的结果是一致的TM机制EtOH调制Cys环受体。这项工作突出了相关的细菌同源物作为有价值的模型系统，用于研究人类受体的离子通道功能，并证明了跨物种的这些通道的模块化。
BackgroundMutagenesis and labeling studies have identified amino acids from the humanα1 glycine receptor (GlyR) extracellular, transmembrane (TM), and intracellular domains in mediating ethanol (EtOH) potentiation. However, limited high‐resolution structural data for physiologically relevant receptors in this Cys‐loop receptor superfamily have made pinpointing the critical amino acids difficult. Homologous ion channels from lower organisms provide conserved models for structural and functional properties of Cys‐loop receptors. We previously demonstrated that a single amino acid variant of the Gloeobacter violaceus ligand‐gated ion channel (GLIC) produced EtOH and anesthetic sensitivity similar to that of GlyRs and provided crystallographic evidence for EtOH binding to GLIC.MethodsWe directly compared EtOH modulation of theα1 GlyR and GLIC to a chimera containing the TM domain from humanα1 GlyRs and the ligand‐binding domain of GLIC using 2‐electrode voltage‐clamp electrophysiology of receptors expressed inXenopus laevisoocytes.ResultsEtOH potentiatedα1 GlyRs in a concentration‐dependent manner in the presence of zinc‐chelating agents, but did not potentiate GLIC at pharmacologically relevant concentrations. The GLIC/GlyR chimera recapitulated the EtOH potentiation of GlyRs, without apparent sensitivity to zinc chelation. For chimera expression in oocytes, it was essential to suppress leakage current by adding 50μM picrotoxin to the media, a technique that may have applications in expression of other ion channels.ConclusionsOur results are consistent with a TM mechanism of EtOH modulation in Cys‐loop receptors. This work highlights the relevance of bacterial homologs as valuable model systems for studying ion channel function of human receptors and demonstrates the modularity of these channels across species.