Binding of PCDH15 to TMC1 for Mechanosensation in the Inner Ear

项目摘要 脊椎动物毛细胞毛束中每个静纤毛的尖端都有一个机械传导 一种将声音和头部运动转化为听觉的复合体 和平衡。TMC 1，一种十次跨膜（TM）蛋白，与 TMEM 16和OSCA离子通道，最近已被确定为成孔离子通道 复合体的亚单位。原钙粘蛋白15（PCDH 15）和钙粘蛋白23（CDH 23）结合以形成一个新的钙粘蛋白。 四聚体，双链蛋白质丝，物理连接蛋白质丝的“尖端连接”， 机械转导复合物的一侧较高，相邻的静纤毛。偏转 发束增加沿着尖端链接的张力，拉动复合物以打开TMC 1。它有 最近通过免疫共沉淀（co-IP）实验表明，PCDH 15和 TMC 1在物理上相互作用，但具体如何作用尚不清楚。 该项目旨在确定TMC 1和PCDH 15中的单个残留物， 结合，并使张力门控TMC 1通道。生物层干涉测量和co-IP 嵌合TMC 1和PCDH 15蛋白的分析将揭示涉及哪些一般区域 在绑定。定点诱变将使结合界面缩小至特定残基。如果 必要的，交联质谱将揭示细胞内的残基间接触， 和细胞外环。如果结合发生在膜内，则经典的色氨酸扫描 将在PCDH 15的单个TM螺旋上采用方案，以找到对于以下过程必需的残基： 与TMC 1的互动最后，非结合性TMC 1突变体和对照将在 TMC 1/TMC 2双敲除小鼠模型，以确定突变体是否正确定位于尖端 但不能介导机械传导。

Project Summary At the tip of each stereocilium in the hair bundle of vertebrate hair cells is a mechanotransduction complex that mediates the conversion of sound and head movements into the senses of hearing and balance, respectively. TMC1, a ten‐pass transmembrane (TM) protein with homology to TMEM16 and OSCA ion channels, has recently been identified as a pore‐forming, ion channel subunit of the complex. Protocadherin 15 (PCDH15) and cadherin 23 (CDH23) bind to form a tetrameric, double‐stranded protein filament, the `tip link' that physically connects the mechanotransduction complex to the side of a taller, neighboring stereocilia. Deflection of the hair bundle increases tension along the tip link, pulling on the complex to open TMC1. It has recently been shown through co‐immunoprecipitation (co‐IP) experiments that PCDH15 and TMC1 physically interact, yet it not known specifically how. This project aims to identify individual residues within TMC1 and PCDH 15 that facilitate binding and that enable tension to gate the TMC1 channel. Biolayer interferometry and co‐IP analysis of chimeric TMC1 and PCDH15 proteins will reveal which general regions are involved in binding. Site‐directed mutagenesis will narrow the binding interface to specific residues. If necessary, cross‐linking mass spectrometry will reveal residue‐to‐residue contacts in intracellular and extracellular loops. If binding occurs within the membrane, a classic tryptophan scanning protocol will be employed on the single TM helix of PCDH 15 to find residues essential for interaction with TMC1. Finally, non‐binding TMC1 mutants and controls will be expressed in a TMC1/TMC2 double knockout mouse model to determine if mutants localize properly to the tips of hair bundle stereocilia but fail to mediate mechanotransduction.