Binding of PCDH15 to TMC1 for Mechanosensation in the Inner Ear

项目摘要 在脊椎动物毛细胞的毛束的每个立体纤毛的顶端有一个机械转导 将声音和头部的运动转化为听觉的复合体 和平衡。TMC1，一种与TMC1同源的十通跨膜(TM)蛋白 TMEM16和OSCA离子通道最近被确定为一种成孔离子通道 复合体的亚单位。原钙粘附素15(PCDH15)和钙粘附素23(CDH23)结合形成 四聚体，双链蛋白细丝，物理上连接 机械转导复合体到较高的、相邻的立体纤毛的一侧。的偏转。 发束增加了沿着尖端链接的张力，拉动复合体打开TMC1。它有 最近通过免疫共沉淀(co-IP)实验表明，PCDH15和 TMC1在物理上相互作用，但还不知道具体是如何作用的。 该项目旨在确定TMC1和PCDH15中的单个残留物，以促进 结合并使张力能够选通TMC1通道。生物膜干涉术与共IP 对嵌合的TMC1和PCDH15蛋白的分析将揭示哪些一般区域涉及 在装订中。定点突变将缩小与特定残基的结合界面。如果 必要的，交联质谱将揭示细胞内残基对残基的接触 和胞外环路。如果膜内发生结合，经典的色氨酸扫描 将在PCDH15的单个TM螺旋上使用协议来寻找对 与TMC1的相互作用。最后，非结合的TMC1突变体和对照将在 TMC1/TMC2双基因敲除小鼠模型，以确定突变体是否正确定位于尖端 毛束立体纤毛，但不能介导机械转导。

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.