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

将编码用半胱氨酸替换Shaker K通道的丝氨酸351的RNA注入爪蟾卵母细胞。在卵母细胞膜上表达Shaker K通道后，将四甲基罗丹明马来酰亚胺结合到电压敏感门控段（S4）的半胱氨酸残基上。在配有超高NA物镜透镜照明的消逝显微镜下观察具有荧光标记的K通道的细胞膜。荧光染料的倏逝场激发允许K通道的单分子图像的可视化。用120 mV的去极化脉冲对细胞膜进行电压钳，诱导单个荧光团图像的荧光强度发生100%变化。当染色介质中含有的染料浓度为完全有效浓度的千分之一时，许多荧光点的强度变化与电压有关，而荧光点的荧光强度变化与电压有关。 ...更多信息 几秒钟后就会变白在这样的条件下，60 mV去极化的电压钳脉冲诱导了大的荧光强度变化，而另一个60 mV的连续去极化没有诱导额外的荧光变化。这一结果表明，在初始半去极化期间，门控段的构象运动发生了量子过程，并且在施加第二半去极化之前，该运动恰好是100%。从这一发现，我们得出结论，每个电压敏感的门控段的K通道之间的休息状态和开放状态的全或无转换。通道的所有四个亚基的门控区段将对离子通道孔的打开和关闭进行表决。蛋白质微机器离子通道可以看作是一个数字到数字的转换器。本研究所采用的光学技术进一步扩展到细胞和组织的改进照明。通过在物镜透镜的后光圈边缘附近引入激光束，激光束以薄片的形状以倾斜的方式照射通过细胞。这种照明方式（狭缝照明）对于细胞对象的光学切片是有用的。缩小窗口宽度并扫描其位置，可以观察到非常高对比度的单分子图像。我们应用这种光学可视化的突触囊泡回收。少

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