Structural and functional analysis of the motor protein prestin using purified protein

使用纯化蛋白对运动蛋白 prestin 进行结构和功能分析

• 批准号: 18390455
• 负责人: WADA Hiroshi
• 金额: $ 10.48万
• 依托单位: Tohoku University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2006
• 资助国家: 日本
• 起止时间: 2006 至 2007
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-18390455/
• 关键词: Otology; Protein; Molecular motor; Prestin; Atomic force microscopy; Purification; Isothermal titration calorimetry; 生物物理; 走査プローブ顕微鏡; パッチクランプ法; 等温滴定型カロリメトリー; 国際研究者交流; アメリカ; 膜タンパク質

Outer hair cells (OHCs), which are sensory cells in the mammalian cochlea, are able to elongate and contract in length in response to changes in memebrane potential. This motility is believed to amplify the motion of the cochlear partition generated by acoustical stimulation. As a result, mammalian hearing is characterized by high sensitivity, wide dynamic range and sharp frequency selectivity. The origin of this motility is associated with conformational change of the motor protein prestin expressed in the lateral wall of OHCs. In the present study, an attempt was made to elucidate the molecular structure and molecular mechanisms of the conformational changes of prestin. First, the plasma membranes of prestin-transfected Chinese hamster ovary (CHO) cells and those of untransfected CHO cells were observed by atomic force microscopy (AFM). More particle-like structures with a diameter of 8-18 nm were found to exist in the plasma membrane of the prestin-transfected CHO cells than in that of the untransfected CHO cells, indicating that the diameter of prestin is 8-12 nm. Second, prestin molecules expressed in the plasma membranes of prestin-transfected CHO cells were labeled with Quantum dots (Qdots), and those dots, about 8 nm in height, were clearly imaged by AFM. Squarish ring-like structures, each with four peaks and one valley at its center, were observed in the vicinity of the Qdots, suggesting that prestin forms a tetramer in the plasma membranes of the prestin-transfected CHO cells. Finally, prestin molecules were purified from prestin-transfected CHO cells. To reveal whether the purified prestin was functional, based on a finding that prestin binds anions, the interaction between purified prestin and chloride was examined by isothermal titration calorimetry. As a result, heat absorption resulting from prestin-chloride interaction was detected, suggesting that the obtained purified prestin was functional.

外毛细胞（OHC）是哺乳动物耳蜗中的感觉细胞，能够响应膜电位的变化而延长和收缩长度。这种运动被认为放大了由声学刺激产生的耳蜗分区的运动。因此，哺乳动物的听觉具有灵敏度高、动态范围宽和频率选择性敏锐的特点。这种运动的起源与 OHC 侧壁表达的运动蛋白 prestin 的构象变化有关。本研究试图阐明prestin的分子结构和构象变化的分子机制。首先，通过原子力显微镜（AFM）观察prestin转染的中国仓鼠卵巢（CHO）细胞和未转染的CHO细胞的质膜。发现prestin转染的CHO细胞质膜中比未转染的CHO细胞存在更多的直径为8-18 nm的颗粒状结构，表明prestin的直径为8-12 nm。其次，在 prestin 转染的 CHO 细胞质膜中表达的 prestin 分子被量子点 (Qdots) 标记，这些点高度约 8 nm，可通过 AFM 清晰成像。在 Qdot 附近观察到方形环状结构，每个结构在其中心有四个峰和一个谷，表明 prestin 在 prestin 转染的 CHO 细胞的质膜中形成四聚体。最后，从 prestin 转染的 CHO 细胞中纯化出 prestin 分子。为了揭示纯化的 prestin 是否具有功能，基于 prestin 结合阴离子的发现，通过等温滴定量热法检查了纯化的 prestin 和氯之间的相互作用。结果，检测到由prestin-氯化物相互作用产生的热吸收，这表明所获得的纯化prestin是有功能的。

项目成果

Immune Atomic Force Microscopy of the Plasma Membrane of Prestin-Transfected Chinese Hamster Ovary Cells Using Quantum Dots

使用量子点对 Prestin 转染的中国仓鼠卵巢细胞质膜进行免疫原子力显微镜检查

• 发表时间: 2008
• 作者: Michio Murakoshi

量子ドットで標識したタンパク質モータプレスチンの原子間力顕微鏡による可視化

量子点标记的蛋白质运动 prestin 的原子力显微镜可视化

• 发表时间: 2008
• 作者: 村越 道生

Purification and isothermal titration calorimetric analysis of the motor protein prestin

运动蛋白 prestin 的纯化和等温滴定量热分析

• 发表时间: 2007
• 作者: Koji Iida

「研究成果報告書概要(和文)」より

摘自《研究结果报告摘要（日文）》

Effects of heat stress on filamentous actin and prestin of outer hair cells in mice

热应激对小鼠外毛细胞丝状肌动蛋白和prestin的影响

• 发表时间: 2007
• 作者: Yoko Kitsunai