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Purification and Functional Analysis of Thermo-sensory channels

热感通道的纯化及功能分析

基本信息

批准号：
14580675
负责人：
NAKAOKA Yasuo
金额：
$ 1.47万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2003
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14580675/
关键词：
Thermosensory Lonic channels Paramecium Membrane fluidity Laurdan Latty acids 温度感覚蛋白精製リゾチーム

项目摘要

From the cell membrane of Paramecium, we tried to isolate and purify the channel proteins whose activity is sensitive to temperature change. Although the purification was, yet incomplete, we found some chemicals that inhibit specifically thermosensory response of Paramecium. The inhibitors were lysozyme, ruthenium-red etc. These inhibitors are polyvalent cations in the neutral solution. From such nature of the inhibitors, following two possibilities are considered for the inhibitory mechanism ; (1) Inhibitors directly bind to the thermosensory channels to block ionic current. (2) Inhibitors indirectly bind to the negatively charged lipids surrounding thermosensory channels, which alters the membrane fluidity and inhibits the activity of channels. In order to test the possibility (2), we constructed the fluorescence microscope system that can measure the membrane fluidity of the living cells stained with fluorescent probe, laurdan. From P. aurelia group, we have already selected each species of high and low thermotolerance. Comparing the membrane fatty acid contents between these species, the high thermotolerant species have a decreased ratio of unsaturated to saturated fatty acids than the low thermotolerant species. Measurements of the membrane fluidity using the fluorescence microscope system showed that the high thermotolerant species had a decreased membrane fluidity from the low thermotolerant species. We will measure rapid changes of membrane fluidity in response to temperature change.

从草履虫细胞膜中分离纯化对温度变化敏感的通道蛋白。虽然纯化不完全，但我们发现了一些特异性抑制草履虫温敏反应的化学物质。在中性溶液中，这些抑制剂是多价阳离子。根据抑制剂的这种性质，认为抑制机制有以下两种可能性：（1）抑制剂直接与热敏通道结合以阻断离子电流。(2)抑制剂间接地与温度敏感通道周围的带负电荷的脂质结合，从而改变膜流动性并抑制通道的活性。为了验证（2）的可能性，我们构建了荧光显微镜系统，用荧光探针laurdan染色，可以测量活细胞的膜流动性。从奥雷利亚类群中，我们已经筛选出了高耐热性和低耐热性的各个种。比较这些物种之间的膜脂肪酸含量，高耐热性的物种有一个比低耐热性的物种的不饱和脂肪酸与饱和脂肪酸的比例下降。利用荧光显微镜系统对膜流动性的测量表明，高耐热性物种的膜流动性比低耐热性物种的膜流动性降低。我们将测量膜流动性随温度变化的快速变化。