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

将编码用半胱氨酸替换 Shaker K 通道的丝氨酸 351 的 RNA 注射到爪蟾卵母细胞中，在卵母细胞膜上表达 Shaker K 通道后，四甲基罗丹明马来酰亚胺与电压敏感门控节段 (S4) 的半胱氨酸残基结合。在配备有超高数值孔径物镜的倏逝显微镜下观察带有荧光标记的 K 通道的细胞膜，用于荧光染料的倏逝场激发，从而实现单个图像的可视化。 K 通道的分子图像。具有 120 mV 去极化脉冲的细胞膜电压钳诱导单个荧光团图像的荧光强度发生 100% 的变化。染色介质中含有的染料浓度为完全有效浓度的千分之一，许多荧光点在几秒钟内显示出电压依赖性强度变化以及量子漂白。在这种条件下，电压钳脉冲。 60 mV 的去极化引起了较大的荧光强度变化，而连续的 60 mV 去极化并没有引起额外的荧光变化。这一结果表明，在最初的半去极化期间，门控部分的构象运动发生了量子过程，并且该运动发生了。在应用去极化的后半部分之前，我们得出结论，K 通道的每个电压敏感门控部分在静息状态之间进行全或无转换。通道的所有四个亚基的门控部分将投票决定离子通道孔的打开和关闭。离子通道，即蛋白质微机器，可以被认为是一个数字到数字的转换器。这项研究进一步扩展到细胞和组织的修改照明，通过在物镜后虹膜边缘附近引入激光束，激光束以倾斜的方式以薄片状照射细胞。的照明（狭缝照明）对于缩小窗口宽度并扫描其位置非常有用，我们将这种光学器件应用于突触囊泡回收的可视化。