Development of Highly Sensitive Bilayr Lipid Membrane Sensors Based on Membrane Proteins

基于膜蛋白的高灵敏双层脂质膜传感器的开发

• 批准号: 04453156
• 负责人: ODASHIMA Kazunori
• 金额: $ 4.48万
• 依托单位: University of Tokyo (1993)Hokkaido University (1992)
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (B)
• 财政年份: 1992
• 资助国家: 日本
• 起止时间: 1992 至 1993
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-04453156/
• 关键词: Biosensor; Ion Channel Protein; Bilayr Lipid Membrane; Glutamate Receptor Ion Channel; NMDA Subtype; Voltage-sensitive Ion Channel; Channel Current; Agonist Selectivity; 平面脂質二分子膜; グルタミン酸作動性イオンチャンネル; 電位作動性ナトリウムイオンチャンネル; クーロメトリー; アフィニティークロマトグラフィ

Fundamental studies aiming at generalization of bilayr lipid membrane sensors, which exploit remarkable signal amplification function of ion channel proteins for highly sensitive sensing of target compounds, were carried out and the following results were obtained.1.Glutamate receptor ion channel protein (GluR), purified from postsynaptic membranes of rat brain, was incorporated into a planar bilayr lipid membrane, and its agonist selectivity was evaluated for several representative agonists under the conditions in which only the NMDA subtype was active. The agonist selectivity based on the total ion permeated through the channels (Ca^<2+> and Na^+) was lower than that of the binding assay, although the order of the selectivity was the same for the two systems. In addition, the agonist selectivity based on the permeation of Ca^<2+> ions was evaluated by thin layr potentiometry using liposomes incorporated with the GluR proteins. A selectivity similar to that for planar bilayr lipid mem … More branes was observed for the NMDA subtype. The present approach based on the transmembrane signals from the GluR proteins (total ions permeated through the membrane) affords a new method for evaluating a biocompatible selectivity, which is different from the binding affinity used so far to evaluate the agonist selectivities of channel proteins.2.Fabrication of a hybrid-type bilayr lipid membrane, incorporated with voltage-sensitive sodium channel proteins (from electric organ of electric eel) with artificial receptor molecules capable of generating guest-induced membrane potential changes, was attempted. Planar bilayr lipid membranes incorporated with the above ion channel protein and valinomycin were prepared. Although the incorporated channel proteins kept ion channel activity, addition of K^+ ion in low concentrations caused a marked decrease in the membrane resistance, indicating the necessity of reinvestigation of the experimental system to make it able to measure the changes in channel currents involving membrane potential changes. Less

本论文对利用离子通道蛋白的信号放大功能对目标化合物进行高灵敏传感的双层类脂膜传感器进行了基础性研究，取得了如下结果：1.将从大鼠脑突触后膜中纯化的谷氨酸受体离子通道蛋白（GluR）整合到平面双层类脂膜中，并在只有NMDA亚型有活性的条件下评价其对几种代表性激动剂的激动剂选择性。基于通过通道渗透的总离子（Ca^2+和Na^+）的激动剂选择性低于结合试验，尽管两种系统的选择性顺序相同。此外，基于Ca^2+离子渗透的激动剂选择性通过薄层电位法使用掺入GluR蛋白的脂质体进行评估。与平面双层脂质体类似的选择性 ...更多信息 膜的NMDA亚型。本方法基于来自GluR蛋白的跨膜信号（渗透通过膜的总离子）提供了一种评价生物相容性选择性的新方法，其不同于迄今为止用于评价通道蛋白的激动剂选择性的结合亲和力。2.混合型双层脂质膜的制备，尝试将电压敏感钠通道蛋白（来自电鳗的电器官）与能够产生客体诱导的膜电位变化的人工受体分子结合。制备了掺入上述离子通道蛋白和缬氨霉素的平面双层脂质膜。尽管掺入的通道蛋白保持了离子通道活性，但加入低浓度的K^+离子会导致膜电阻显著降低，这表明有必要重新研究实验系统，使其能够测量涉及膜电位变化的通道电流变化。少

项目成果

K.Tohda, K.Odashima, M.Sugawara, and Y.Umezawa: ""Chemical Sensing Based on Transmembrane Signals"" Maku. 19(2). 122-132 (1994)

K.Tohda、K.Odashima、M.Sugarara 和 Y.Umezawa：“基于跨膜信号的化学传感”Maku。

小田嶋 和徳: "分子、認識界面を基礎とするセンシング化学" 電気化学および工業物理化学. 60・1. 14-21 (1992)

小田岛一典：“基于分子和识别界面的传感化学”电化学和工业物理化学60・1（1992）。

Y.Hatanaka: "An Improved Synthesis of 4-[3-Trifluoromethyl)-3H-diazirin-3-yl]benzoic acid for Photoaffinity Labeling" Heterocycles. 35. 997-1004 (1993)

Y.Hatanaka：“用于光亲和标记的 4-[3-三氟甲基）-3H-二氮杂环-3-基]苯甲酸的改进合成”杂环。

K.Odashima: "Potentiometric Discrimination of Organic Amines by a Liquid Membrane Electrode Based on a Lipophilic Hexaester of Calix[6]arene" Analytical Chemistry. 65. (1993)

K.Odashima：“基于杯[6]芳烃的亲脂性六酯的液膜电极对有机胺的电位鉴别”分析化学。

E.K.Michaelis, M.L.Michaelis, K.N.Kumar, N.Tilakaratne, D.B.Joseph, P.S.Johnson, K.K.Babcock, G.L.Aistrup, R.L.Schowen, H.Minami, M.Sugawara, K.Odashima, and Y.Umezawa: ""Purification, Reconstitution, and Cloning of an NMDA Receptor-Ion Channel Complex fr

E.K.Michaelis、M.L.Michaelis、K.N.Kumar、N.Tilakaratne、D.B.Joseph、P.S.Johnson、K.K.Babcock、G.L.Aistrup、R.L.Schowen、H.Minami、M.Sukawara、K.Odashima 和 Y.Umezawa：“”纯化、重构