动物的听觉依赖于耳蜗内的毛细胞。毛细胞有着独特的机械—电转导机制，通过顶连接（tip-link）机械门控静纤毛顶端的机械—电转导（Mechano-electrical transduction, MET）离子通道，将声音振动转化为生物电信号，引起听神经的兴奋和冲动，进而传至大脑产生听觉。PCDH15（protocadherin15）位于毛细胞顶连接的底端，是机械门控结构的重要组分。对PCDH15结构和功能的研究将有助于揭示毛细胞机械—电转导的机制。本论文着眼于PCDH15的胞外近膜区一段被称为EL（extracellular linker）的结构域，其具备“βαββαβ”状的拓扑结构，但在毛细胞中的功能仍然有待确定。 本论文中，我们首先对耳蜗毛细胞电转和钙成像实验体系进行了优化，为论文工作提供了方法学基础。随后，通过在培养的耳蜗毛细胞上外源导入不同的EL结构域缺失的PCDH15质粒，观察PCDH15在毛细胞胞体和静纤毛上的表达，以及Ames waltzer-3j（av3j）小鼠毛细胞机械转导活性的恢复情况，我们发现EL外围的一个α螺旋（H1）缺失使PCDH15无法定位到毛细胞静纤毛，但不影响PCDH15向质膜的转运；另一个α螺旋（H2）缺失增加毛细胞机械转导的强度，但不通过增加毛细胞静纤毛数量，也不通过影响PCDH15与TMIE、TMHS及TMC1/2等MET通道复合物的蛋白相互作用。紧接着我们进一步寻找和研究了EL的进化保守同源结构对于蛋白在毛细胞定位和功能的意义；我们通过结构预测发现CDH23和PCDH24的胞外近膜结构域（MAD）与EL有相似的结构，且CDH23的MAD对于CDH23和PCDH15定位到静纤毛有调控作用。此外，我们还尝试对Pcdh15移码突变引起的av3j耳聋小鼠进行基因治疗；利用基于Cas9的基因编辑技术，在体外和体内系统分别使73.5%和超过78.3%的毛细胞恢复了机械敏感性，且降低了av3j小鼠听力的阈值。最后，基于所建立的毛细胞电转与钙成像体系方法学，我们尝试了从耳蜗差异性表达基因中筛选毛细胞机械—电转导相关基因。 综上，本论文中我们基于前人报道的EL的蛋白结构，首次在听觉毛细胞中对EL结构域的功能进行了探究。我们的工作表明EL参与了PCDH15在毛细胞的定位以及毛细胞的机械—电转导过程。我们的工作为进一步研究顶连接的形成机制，以及PCDH15门控毛细胞MET通道的机制拓展了思路。
The sense of hearing relies on the hair cells that locate inside of the cochlea. The hair cells endow with special mechano-electrical transduction machinery. By utilizing the tip-links mechanically gate the mechano-electrical transduction (MET) ion channels at the tip of the stereocilia, the hair cells convert sound vibrations into biological electrical signals, which are then sent into brain via auditory nerve to generate the sense of hearing. Protocadherin 15 (PCDH15), the protein that made up the lower portion of tip-link, is a critical component of the mechanotransduction complex. Insights into the structure and functionality of PCDH15 are essential for understanding the MET channel mechanism, which is, for now, not fully understood. This dissertation focused on a domain in the membrane adjacent region of PCDH15 named EL (extracellular linker). Previous studies revealed that EL shows a topology of “βαββαβ”, but its function remains largely unknown. In this dissertation, we first improved the experimental systems for hair cell injectoporation and calcium imaging. These improvements provided a methodological basis for our following studies. Secondly, EL domain-truncated PCDH15 plasmids were imported into cultured cochlear hair cells to observe the expression of PCDH15 in cell body and stereocilia, as well as the recovery of mechanical sensitivity of hair cells from Ames waltzer-3j (av3j) mice. We found that deletion of an α helix (H1) in the periphery of EL prevented PCDH15 from locating to the hair cell stereocilia, but did not affect the transport of PCDH15 to the plasma membrane. Deletion of another α helix (H2) increased the intensity of hair cell MET, but not by increasing the number of hair cell stereocilia or by affecting the protein-protein interactions between PCDH15 and previously identified MET ion channel components including TMIE, TMHS, and TMC1/2. We further identified the evolutionarily conserved homologous structures of EL and explored the meaning of these structures for protein localization and function in hair cells. We predicted that the extracellular domains (MADs) of CDH23 and PCDH24 were similar to that of EL, and the MAD of CDH23 also could regulate the targeting of CDH23 and PCDH15 to stereocilia. In addition, gene therapy was explored on Pcdh15 frame-shifting mutation-induced deafness in av3j mice. Using cas9-based gene editing, mechanical sensitivity was restored in 73.5% hair cells in vitro and over 78.3% in vivo, respectively. The gene therapy also reduced the threshold of hearing in av3j mouse. Finally, with our improved experimental system, we tried to screen genes related to hair cell MET from differentially expressed genes in the cochlea using hair cell injectoporation and calcium imaging systems. In conclusion, based on the known protein structure of EL reported previously, we investigated, for the first time, the function of EL domain in auditory hair cells. Our study indicated that EL mediated the localization of PCDH15 in hair cells, and might be involved in hair cell MET. Besides, this study provides new insights for further researching the mechanisms underlying tip-link formation and how PCDH15 gated the MET ion channels of the hair cells.
