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

利用离子通道蛋白显著的信号放大功能，实现对目标化合物的高灵敏度传感，针对双膜脂膜传感器的推广进行了基础研究，获得了以下结果：从大鼠脑突触后膜中纯化出谷氨酸受体离子通道蛋白（GluR），将其整合到平面双层脂质膜中，在仅NMDA亚型有活性的条件下，对几种代表性激动剂的选择性进行了评价。尽管两种系统的选择性顺序相同，但基于通道中渗透的总离子（Ca^<2+>和Na^+）的激动剂选择性低于结合试验。此外，基于Ca^<2+ b>离子的渗透，通过脂质体与GluR蛋白结合的薄层电位法评估了激动剂的选择性。NMDA亚型具有与平面双层脂质膜相似的选择性。目前基于GluR蛋白（通过膜渗透的总离子）的跨膜信号的方法，为评价通道蛋白的生物相容性选择性提供了一种新的方法，它不同于目前用来评价通道蛋白的结合亲和力。本文尝试制备一种混合型双膜脂质膜，将电压敏感钠通道蛋白（来自电鳗的电器官）与人工受体分子结合，能够产生客诱导的膜电位变化。制备了含有上述离子通道蛋白和缬霉素的平面双层脂质膜。虽然加入的通道蛋白保持了离子通道的活性，但低浓度的K^+离子的加入导致了膜阻力的显著降低，这表明有必要对实验系统进行重新研究，使其能够测量涉及膜电位变化的通道电流的变化。少

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