Structure-function linkage of ion channels and transporters : studies with new optical technology, optical tweezers and evanescent light microscopy.

将最新的光学技术应用于研究活细胞离子通道和转运体的动力学是很有必要的。在本研究中，我们尝试在细胞生理学领域中使用激光光镊子和消逝光。我们用100 mW的Ar-Kr激光提高了镊子的能力，这种镊子可以在培养的大鼠神经元生长锥体表面固定和移动乳胶珠长达1um。在高分辨率视频增强差示干涉显微镜中引入了渐散光照明。将培养在玻片上的神经元生长锥和胞体用荧光染料DiI染色，并在内全反射消逝光显微镜下观察。瞬息万变的图像显示，生长锥体和有限的胞体附着在培养玻璃板上。同时，使用Ohtsu等人研制的照明模式消逝光显微镜，我们获得了…在分析伸展激活的离子通道行为的过程中，克隆了ClC-2，并对非洲爪哇卵母细胞表达的ClC-2电流进行了表征。结论：ClC-2电流对卵母细胞体积有调节作用。此外，还克隆了具有延伸活性的ClC-2通道调节蛋白，并对其结构进行了测定。我们正试图通过制备一种针对这种蛋白质的荧光偶联抗体来利用逝去的光来可视化这种蛋白质。同时，我们可以记录机械刺激作用于培养的DRG神经元生长锥的内向电流。用急性分离的海马神经元作为生物检测器检测生长锥中谷氨酸的释放，并进行全细胞膜片钳记录。这种释放依赖于Ca~(2+)和gt；，并被omega-agatoxin所阻断。用毒素对培养的神经元进行预处理，然后与毒素抗体孵育，再用金标记抗体进行染色。由于在扫描和透射电子显微镜下观察到免疫金粒聚集在生长锥体的膜上，因此推测omega-agatoxin敏感的Ca~(2+)&gt~(2+)通道在生长锥体上有免疫定位。较少

It is necessary to apply recent advance in optical technology to studying the dynamics of ion channels and transporter of living cells. In the present study, we have attempted to employ laser optical tweezers and evanescent light in the field of cellular physiology. We improved the ability of the tweezers by using 100 mW Ar-Kr laser beam ; the tweezers could hold and move latex beads up to 1mum on the surface of growth cones of rat cultured neurons.Evanescent light illumination was introduced into a high resolution video-enhanced differential interference contrast microscope. Growth cones and soma of neurons which were cultured on glass plates were stained with a fluorescent dye, DiI,and were observed with an internal total reflection evanescent light microscope. The evanescent images showed that growth cones and only limited areas of soma attached to the culture glass plate. At the same time, using an illumination mode evanescent light microscope developed by Ohtsu et al, we obtained … More images of bundles of tubulin or neurofilament in neurite or growth cone regions.In the course of analyzing behavior of ion channels activated by extension, ClC-2 was cloned and ClC-2 currents expressed in Xenopus oocytes were characterized. And it is concluded that ClC-2 currents may regulate the oocyte volume. Furthermore an extension-actived ClC-2 channel-modulating protein was cloned and its structure was determined. We are trying to visualize this protein using evanescent light by preparing a fluorescence-conjugated antibody to the protein. At the same time, we could record inward currents in response to mechanical stimulation applied to growth cones of cultured DRG neurons.Glutamate release from growth cones was detected by using an acutely dissociated hippocampal neuron as a biodetector from which whole cell patch clamp recordings were made. The release was dependent on Ca^<2+> and was abolished by omega-agatoxin. Cultured neurons were pretreated with the toxin, then incubated with the antibody to the toxin and further stained with gold-conjugated antibody. Because clusters of immuno-gold particles were observed over the membrane of growth cones by means of scanning and transmission electron microscope, it is suggested omega-agatoxin-sensitive Ca^<2+> channels were immunologically localized on growth cones. Less