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

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伸长和收缩。这种能动性被认为放大了由声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强的特点。这种运动的起源与外毛细胞侧壁表达的运动蛋白普雷斯廷的构象变化有关。本研究试图阐明普雷斯廷的分子结构和构象变化的分子机制。首先，用原子力显微镜（AFM）观察转染prestin的中国仓鼠卵巢（CHO）细胞和未转染CHO细胞的质膜。转染Prestin基因的CHO细胞质膜上出现了更多直径为8-18 nm的颗粒状结构，表明普雷斯廷的直径为8-12 nm。第二，用量子点（Qdots）标记在prestin转染的CHO细胞的质膜中表达的普雷斯廷分子，并且通过AFM清楚地成像这些点，约8 nm高。在Qdots附近观察到方形环状结构，每个结构在其中心具有四个峰和一个谷，这表明普雷斯廷在prestin转染的CHO细胞的质膜中形成四聚体。最后，从prestin转染的CHO细胞中纯化普雷斯廷分子。为了揭示纯化的普雷斯廷是否是功能性的，基于普雷斯廷结合阴离子的发现，通过等温滴定量热法检查纯化的普雷斯廷和氯化物之间的相互作用。结果，检测到由prestin-氯化物相互作用引起的热吸收，表明所获得的纯化的普雷斯廷是功能性的。

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.