Purification and Functional Analysis of Thermo-sensory channels

本研究试图从草履虫细胞膜中分离纯化其活性对温度变化敏感的通道蛋白。虽然纯化还不完全，但我们发现了一些化学物质可以特异性地抑制草履虫的热敏反应。缓蚀剂有溶菌酶、钌红等，这些缓蚀剂在中性溶液中为多价阳离子。从抑制剂的这种性质出发，考虑了以下两种可能的抑制机制：(1)抑制剂直接与热敏通道结合，以阻断离子电流。(2)抑制剂间接与温敏通道周围带负电荷的脂类结合，改变膜流动性，抑制通道活性。为了验证(2)的可能性，我们构建了荧光显微镜系统，可以测量用荧光探针Laurdan染色的活细胞的膜流动性。从金龟子群中，我们已经筛选出了耐热性高和耐低温性的各个物种。比较这些物种之间的膜脂肪酸含量，耐热性高的物种的不饱和脂肪酸与饱和脂肪酸的比率低于耐热性低的物种。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。

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.