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

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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