Identification of Binding Partners of the Hair‐Cell Protein Otoferlin Using Co‐Immunoprecipitation and Proximity‐Dependent Labelling Techniques

Abstract

Otoferlin is a calcium-sensitive multi-C2-domain transmembrane protein which localizes to auditory hair cell synapses and is associated with congenital deafness. Although the role of otoferlin in synaptic vesicle exocytosis in cochlear hair cells is widely accepted, the exact function(s) of the protein are poorly understood. This lack of understanding both prohibits formation of mechanistic models describing the encoding of sound, and prevents development of therapeutic treatments for deafness. Further, a lack of traditional synaptic vesicle proteins in hair cells suggests a fundamentally different method for synaptic vesicle exocytosis in these cells. In this study, we used a combination of co-immunoprecipitation and proximity-labelling methods to identify otoferlin binding partners that may participate in hair cell exocytosis. As co-immunoprecipitation involves lysing the cell, and lacks the ability to capture weak and transient interactions, an in-cell proximity-dependent labelling assay was used to find protein partners. Analysis of samples using mass spectroscopy allowed us to comprehensively characterize a molecular interaction network for otoferlin. This otoferlin binding network leads us to propose a novel molecular-level mechanism for neurotransmission at hair cell synapses.