Deciphering the molecular function of otoferlin in synaptic vesicle replenishment in inner ear sensory hair cells

耳铁蛋白在内耳的感觉毛细胞中表达，属于铁蛋白家族。Ferlin蛋白共享由串联C2结构域和单个C-末端跨膜区段组成的保守结构，并且在肌肉和上皮细胞中功能性地参与Ca 2+触发的膜融合过程。Otoferlin在隐性形式的严重人类语前听力损失DFNB 9中是有缺陷的，并且otoferlin缺陷小鼠具有由感觉毛细胞的带状突触处的突触囊泡的Ca 2+依赖性补充减少引起的严重听力损伤。为了破译这一过程背后的otoferlin的分子功能，本研究计划的目标是基于蛋白质组学的鉴定和功能表征的otoferlin相互作用的合作伙伴。为了达到这一目标，我将进行免疫共沉淀的otoferlin从毛细胞的听觉和前庭器官的鸡胚耳朵。使用超灵敏的质谱，免疫沉淀蛋白将被确定和候选蛋白选择根据Ca 2+信号，囊泡循环，或膜融合的潜在作用。我在一个试点实验中发现了AHNAK 1等候选基因，将分三步进行进一步分析：i）用共聚焦免疫荧光和电子显微镜测定它们在毛细胞中的亚细胞定位，ii）重组表达的otoferlin和相互作用配偶体的GST-下拉测定（如AHNAK 1），以证明物理相互作用并确定特异性结合位点，和iii）使用适当的敲除小鼠来评估新的耳传递蛋白相互作用配偶体在突触囊泡补充中的生理相关性。我设想，解开otoferlin功能的分子网络，将大大推进突触囊泡如何在毛细胞中移动和胞吐的基础知识，以及这些过程如何构成听力和前庭功能的基础。

Otoferlin is expressed in sensory hair cells of the inner ear and belongs to the ferlin protein family. Ferlins share a conserved architecture consisting of tandem C2 domains, and a single C-terminal transmembrane segment and are functionally involved in Ca2+-triggered membrane fusion processes in muscle and epithelial cells. Otoferlin is defective in a recessive form of profound human prelingual hearing loss DFNB9, and otoferlin-deficient mice have a profound hearing impairment caused by reduced Ca2+-dependent replenishment of synaptic vesicles at the ribbon synapses of sensory hair cells. To decipher the molecular function of otoferlin underlying this process, the goal of this research proposal is a proteomic based identification and functional characterization of otoferlin interaction partners. To reach this goal, I will conduct co-immunoprecipitations of otoferlin from hair cells of the auditory and vestibular organs of embryonic chicken ears. Using ultrasensitive mass spectrometry, immunoprecipitated proteins will be identified and candidate proteins selected according to a potential role in Ca2+-signaling, vesicle cycling, or membrane fusion. Candidates such as AHNAK1, which I identified in a pilot-experiment, will be further analyzed in three steps: i) determination of their subcellular localization(s) in hair cells with confocal immuno-fluorescence and electron microscopy, ii) GST-pulldown assays of recombinantly expressed otoferlin and interaction partners (such as AHNAK1) to prove a physical interaction and to determine specific binding sites, and iii) usage of appropriate knock-out mice to assess a physiological relevance of the novel otoferlin interaction partner in synaptic vesicle replenishment. I envision that unravelling the molecular network in which otoferlin functions, will substantially advance fundamental knowledge of how synaptic vesicles are moved and exocytosed in hair cells, and how these processes underlie hearing and vestibular functions.