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

离子通道和转运蛋白的结构-功能联系：利用新光学技术、光镊和倏逝光显微镜进行研究。

• 批准号: 07457010
• 负责人: KATAYAMA Yoshifumi
• 金额: $ 4.74万
• 依托单位: Tokyo Medical and Dental University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1997
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07457010/
• 关键词: Optical tweezers; Evanescent light; Membrane protein; Ion channel; Imaging; Cloning; Evanescent light microscopy; Optical technology; カルシウムチャネル; クロライドチャネル; メカノレセプター; 神経成長円錐; トランスポータ; 微分干渉顕微鏡; 近接場顕微鏡

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

有必要应用光学技术的最新进展来研究活细胞离子通道和转运蛋白的动力学。在本研究中，我们尝试在细胞生理学领域使用激光光镊和倏逝光。我们通过使用100 mW Ar-Kr激光束提高了镊子的能力；镊子可以在大鼠培养的神经元的生长锥表面上固定和移动乳胶珠达1μm。将倏逝光照明引入高分辨率视频增强微分干涉相差显微镜中。将玻璃板上培养的神经元的生长锥和体细胞用荧光染料DiI染色，并用内全反射倏逝光显微镜观察。转瞬即逝的图像显示，生长锥和仅有有限区域的体细胞附着在培养玻璃板上。同时，使用 Ohtsu 等人开发的照明模式倏逝光显微镜，我们获得了神经突或生长锥区域中的微管蛋白或神经丝束的图像。在分析延伸激活的离子通道的行为过程中，克隆了 ClC-2，并对非洲爪蟾卵母细胞中表达的 ClC-2 电流进行了表征。并得出ClC-2电流可能调节卵母细胞体积的结论。此外，克隆了延伸激活的ClC-2通道调节蛋白并确定了其结构。我们正在尝试通过制备该蛋白质的荧光偶联抗体，利用倏逝光来可视化该蛋白质。同时，我们可以记录对培养的 DRG 神经元生长锥施加机械刺激的内向电流。通过使用急性分离的海马神经元作为生物探测器来检测生长锥的谷氨酸释放，并从中进行全细胞膜片钳记录。该释放依赖于Ca 2+ 并被omega-agatoxin 消除。用毒素预处理培养的神经元，然后与毒素抗体一起孵育，并进一步用金缀合抗体染色。因为通过扫描和透射电子显微镜在生长锥的膜上观察到免疫金颗粒簇，这表明omega-雷加毒素敏感的Ca 2+ 通道在免疫学上定位于生长锥上。较少的

项目成果

Morita,K.and Katayama,Y.: "Tetraethylammonium-sensitive calcium-sensitive pottasium current in a subclass of the bullfrog dorsal root ganglion cells." Neuroscience Letters. 215. 193-196 (1996)

Morita,K. 和 Katayama,Y.：“牛蛙背根神经节细胞亚类中的四乙铵敏感钙敏感钾电流。”

Tatsumi H,Tsuii S,Anglade IP,Motelica-Heino I,Soeda H,Katayama Y: "Synthesis storage and release of acetylcholine at and from growth cones of rat central cholinergic neurons in culture." Neuroscience Letters. 202. 1-4 (1995)

Tatsumi H、Tsuii S、Anglade IP、Motelica-Heino I、Soeda H、Katayama Y：“培养物中大鼠中枢胆碱能神经元生长锥中乙酰胆碱的合成储存和释放。”

Uma Maheswari R et al.(5 authors): "Cbservation of subcellular structures of neurons by an illumination mode near-field optical microscope under・・・" Optical Review. 3. 463-467 (1996)

Uma Maheswari R 等人（5 位作者）：“在……下通过照明模式近场光学显微镜观察神经元的亚细胞结构”光学评论 3. 463-467 (1996)。

Ono A,Tatsumi H,Yamamoto K,Katayama Y: "Human B lymphocytes respond to Epstein-Barr virus with an increase in intracellular Ca^<2+> concentration." Bull. Tokyo Medical & Dental Univ.42. 9-18 (1995)

Ono A、Tatsumi H、Yamamoto K、Katayama Y：“人类 B 淋巴细胞对 Epstein-Barr 病毒做出反应，细胞内 Ca^2 浓度增加。”

Tatsumi H, Sokabe M, Katayama Y.: "Observation of neuronal growth cones with evanscent light microscope." Neuroscience Research. Suppl 21. S142 (1997)

Tatsumi H、Sokabe M、Katayama Y.：“用倏逝光显微镜观察神经元生长锥。”