Horani Suzzane, Stater Evan P, Corringer Pierre-Jean, Trudell James R, Harris R Adron, Howard Rebecca J
Waggoner Center for Alcohol and Addiction Research, The University of Texas at Austin, Austin, Texas.
Chemistry Department , Skidmore College, Saratoga Springs, New York.
Alcohol Clin Exp Res. 2015 Jun;39(6):962-8. doi: 10.1111/acer.12735. Epub 2015 May 14.
Mutagenesis and labeling studies have identified amino acids from the human α1 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.
We directly compared EtOH modulation of the α1 GlyR and GLIC to a chimera containing the TM domain from human α1 GlyRs and the ligand-binding domain of GLIC using 2-electrode voltage-clamp electrophysiology of receptors expressed in Xenopus laevis oocytes.
EtOH potentiated α1 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 50 μM picrotoxin to the media, a technique that may have applications in expression of other ion channels.
Our 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.
诱变和标记研究已确定人类α1甘氨酸受体（GlyR）细胞外、跨膜（TM）和细胞内结构域中的氨基酸在介导乙醇（EtOH）增强作用方面发挥作用。然而，该半胱氨酸环受体超家族中生理相关受体的高分辨率结构数据有限，使得确定关键氨基酸变得困难。来自低等生物的同源离子通道为半胱氨酸环受体的结构和功能特性提供了保守模型。我们之前证明，蓝细菌紫质配体门控离子通道（GLIC）的单个氨基酸变体产生的乙醇和麻醉敏感性与甘氨酸受体相似，并提供了乙醇与GLIC结合的晶体学证据。
我们使用非洲爪蟾卵母细胞中表达的受体的双电极电压钳电生理学，将α1甘氨酸受体和GLIC的乙醇调节作用与包含人类α1甘氨酸受体TM结构域和GLIC配体结合结构域的嵌合体进行了直接比较。
在存在锌螯合剂的情况下，乙醇以浓度依赖性方式增强α1甘氨酸受体，但在药理学相关浓度下不增强GLIC。GLIC/甘氨酸受体嵌合体重现甘氨酸受体的乙醇增强作用，对锌螯合无明显敏感性。对于嵌合体在卵母细胞中的表达，通过向培养基中添加50μM印防己毒素来抑制漏电流至关重要，该技术可能在其他离子通道的表达中具有应用价值。
我们的结果与半胱氨酸环受体中乙醇调节的TM机制一致。这项工作强调了细菌同源物作为研究人类受体离子通道功能有价值模型系统的相关性，并证明了这些通道在物种间的模块化。
