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

外毛细胞是哺乳动物耳蜗中的感觉细胞，能够随着膜电位的变化而伸长和收缩。这种运动性被认为放大了由声刺激产生的耳蜗区的运动。因此，哺乳动物的听力具有灵敏度高、动态范围宽、频率选择性强等特点。这种运动的起源与毛细胞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构象变化的分子结构和分子机制。首先，用原子力显微镜(AFM)观察了转prestin基因的中国仓鼠卵巢(CHO)细胞和未转染CHO细胞的细胞膜。与未转染的CHO细胞相比，转入prestin的CHO细胞质膜中有较多的直径为8~18 nm的颗粒状结构，表明prestin的直径为8~12 nm。其次，用量子点(Quantum dots，Qdots)标记表达于细胞膜上的prestin分子，这些量子点的高度约为8 nm，并用原子力显微镜(AFM)清晰地成像。在Qdots附近观察到四峰一谷的方形环状结构，提示prestin在转染prestin的CHO细胞质膜中形成四聚体。最后，从转染prestin的CHO细胞中纯化了prestin分子。为了揭示纯化的Prestin是否具有功能，基于Prestin与阴离子结合的发现，用等温滴定量热法研究了纯化的Prestin与氯化物之间的相互作用。结果检测到Prestin与氯离子相互作用产生的热吸收，表明所获得的纯化Prestin是有功能的。

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.