Deciphering the molecular function of otoferlin in synaptic vesicle replenishment in inner ear sensory hair cells
Final Report Abstract
At the hair cell afferent fiber synapse, auditory information is converted into electrical signals and transmitted through the auditory nerve to the central nervous system (CNS), so that we can hear. This performance involves unique properties of the inner hair cell synapse and any interference with this process results in hearing loss. Thus, understanding the molecular mechanisms underlying synaptic transmission at inner hair cell synapses is essential for understanding disease processes. The mechanism specializing the hair cell synapse remains unknown but otoferlin, a protein absent from all ribbon containing cells but for hair cells, has been implicated in this process. Otoferlin is a 230 kDa integral transmembrane protein, containing six C2 domains that are implicated in calcium and phospholipid binding. Dysfunction of otoferlin is responsible for an auditory neuropathy in humans, a type of hearing loss, which occurs when sound is not transmitted properly from the inner hair cells to the brain. Otoferlin was shown to play a role in inner hair cell synaptic vesicle fusion, endocytosis, and fast synaptic vesicle replenishment, however, the molecular mechanism underlying these processes is largely unknown. Performing immunoprecipitation of otoferlin from chicken vestibular hair cells and high resolution mass spectrometry, otoferlin was found to be phosphorylated and the calcium/calmodulin dependent protein kinase CaMKII delta was identified as potential interaction partner. A physical contact of both proteins was confirmed by pull-down experiments from a recombinant system where CaMKII delta was co-purified with otoferlin from HEK293 cells. In vitro phosphorylation assays with heterologously expressed mouse otoferlin and recombinant CaMKII delta further confirmed otoferlin phosphorylation by this kinase. Mass spectrometric analysis of in vitro phosphorylated otoferlin identified ten specific phosphorylation sites, of which six are located within otoferlin C2 domains and seven are conserved amongst otoferlin species (human, rat, mouse, zebrafish and chicken). Three of then sites, that are located outside of a C2 domain, are only found in rat and mouse or human otoferlin. All in all, this suggests that otoferlin function is regulated by phopsphorylation, possibly modulating calcium and phospholipid binding in the C2 domains or conferring stability to the protein. Future studies will be performed to probe a physiological function of CaMKII delta in mammalian inner hair cells and to examine if and how otoferlin phosphorylation modulates synaptic transmission at the afferent hair cell synapse.
