Identification of Proteins that Interact with Otoferlin to build a ‘Otoferlin Interactome’

Abstract

Otoferlin is a C2‐domain transmembrane vesicular protein that has been associated with congenital deafness. It is majorly responsible for facilitating synaptic vesicle exocytosis in cochlear hair cells. Synaptotagmin and Otoferlin have previously been compared to one another for their calcium sensor functions, but hair cells exocytosis is not as well‐understood as neuronal exocytosis. In case of hair cell synapses, we still lack complete knowledge of the SNARE proteins or their equivalent that aid in the fusion of vesicle membranes to the presynaptic membranes, thereby leading to exocytosis. Currently, a major bottleneck in our understanding of Otoferlin’s role in hearing and deafness can be attributed to the lack of knowledge about its mechanism of functioning. Using brain extracts from mouse, we conducted coimmunoprecipitation experiments to find interaction partners for Otoferlin so as to build a Otoferlin interactome, that can shed some light on the mechanism by which hair cell exocytosis actually occurs. The proteins interacting with Otoferlin were identified by Mass spectroscopy and consisted largely of neuronal proteins that share functions with SNARE proteins or had other neurotransmission‐related functions. We use Human Embryonic Kidney (HEK) cells and a zebrafish model to validate the direct interactors of Otoferlin and conduct further assays to determine their role in hair cell exocytosis and/or endocytosis. This knowledge of Otoferlin interactome leads us to a better understanding of the mechanism whereby neurotransmission is carried out in the inner hair cells.Support or Funding Information1R01DC014588 (C.P. Johnson) 4/2016‐3/2021 NIDCD (NIH). In vivo and in vitro studies of the deafness associated protein otoferlin