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Dynamic study of ion channels by objective-lens-illuminating evanescence microscopy

物镜照明倏逝显微镜对离子通道的动态研究

基本信息

批准号：
14370010
负责人：
TERAKAWA Susumu
金额：
$ 8.9万
依托单位：
Hamamatsu University School of Medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2004
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14370010/
关键词：
Single channel visualization K channel Voltage sensor Evanescence microscope Single molecule Ion channel pore Slit scanning microscope Gating current 電圧依存性膜電位固定実験全反射照明溶媒効果イオンコンダクタンス

项目摘要

RNA encoding replacement of serine 351 of the Shaker K channel with cystein was injected into the Xenopus oocyte. After expression of the Shaker K channel on the oocyte membrane, tetramethyl rhodamine maleiniide was bound to the cystein residue of the voltage sensitive gating segment (S4). The cell membrane with fluorescently labeled K channel observed under the evanescence microscope equipped with an ultra high NA objective lens for illumination. Evanescent field excitation of the fluorescent dye allowed visualization of single molecule image of the K channel. The voltage clamp of the cell membrane with a depolarizing pulse of 120 mV induced 100% change in fluorescence intensity of the single fluorophore image. A photo bleaching of the quantal manner was occasionally observed in a fluorescence spot.When the staining medium contained the dye at a concentration one thousandth of fully effective one, many fluorescent spots showed the voltage dependent intensity change as well as the quan … More tal bleach in a few seconds. Under such conditions, a voltage clamp pulse of 60 mV depolarization induced a large fluorescence intensity change, whereas successive depolarization of another 60 mV did not induce additional fluorescence change. This result suggested that a quantum process takes place in the conformational movement of gating segment during the initial half depolarization and that the movement happened to be 100% before the second half of depolarization was applied. From this finding, we concluded that each voltage sensitive gating segment of the K channel makes all-or-none transition between the resting state and opening state. The gating segments of all four subunits of a channel would make a vote for the opening and closing of the ion channel pore. Ion channel, the protein micromachine, can be considered as a digital to digital converter.The optical technique adopted for this investigation was further extended to modified illumination of the cells and tissue. By introducing a laser beam near the margin of back iris of objective lens, a laser beam was shone through the cells in an oblique manner in a shape of thin sheet This way of illumination (slit illumination) was useful for optical sectioning of cellular objects. Narrowing the window width and scanning its position, the single molecule image was observed at very high contrast. We applied this optics to visualization of synaptic vesicle recycling. Less

将编码Shaker K通道丝氨酸351位半胱氨酸的RNA注射到非洲爪哇卵母细胞中。在卵母细胞膜上表达Shaker K通道后，四甲基罗丹明马来酸与电压敏感门控片段的半胱氨酸残基结合(S4)。在配备超高NA物镜照明的消逝显微镜下观察到带有荧光标记的K通道的细胞膜。荧光染料的逝去场激发使K通道的单分子图像可视化。在120 mV的去极化脉冲作用下，细胞膜上的电压钳使单个荧光团的荧光强度发生了100%的变化。在荧光点中偶尔观察到量子方式的光漂白，当染色介质中含有全效染料的千分之一时，许多荧光点表现出电压依赖的强度变化以及量子…几秒钟后会有更多的TAL漂白剂。在此条件下，60 mV的电压钳脉冲退极化引起较大的荧光强度变化，而另一个60 mV的电压钳脉冲不会引起额外的荧光变化。这一结果表明，在初始半退极化过程中，门控段的构象运动发生了量子过程，在后半退极化之前，该运动恰好是100%。根据这一发现，我们得出结论，K通道的每个电压敏感门控段在静息状态和开放状态之间进行全有或全无转换。通道的所有四个亚单位的门控段将投票支持离子通道孔的打开和关闭。离子通道，即蛋白质微机械，可以被看作是一个数字到数字的转换器。本研究采用的光学技术进一步扩展到细胞和组织的改进照明。通过在物镜后虹膜边缘附近引入一束激光，激光束以倾斜的方式以薄片的形状穿过细胞，这种照明方式(狭缝照明)有助于对蜂窝状物体进行光学切片。缩小窗口宽度并扫描其位置，以很高的对比度观察到单分子图像。我们将这种光学技术应用于突触囊泡循环的可视化研究。较少