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

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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.