Structure and function of otoferlin, a synaptic protein of sensory hair cells essential for hearing

Hearing relies upon speedy synaptic transmission of sound information from inner hair cells (IHCs) to spiral ganglion neurons. To accomplish this, IHCs use a sophisticated presynaptic machinery including the multi-C₂ domain protein otoferlin that is affected by human deafness mutations. Otoferlin is essential for IHC exocytosis, but how it binds Ca²⁺ and the target membrane to serve synaptic vesicle (SV) tethering, docking, and fusion remained unclear. Here, we obtained cryo-electron microscopy structures of otoferlin and employed molecular dynamics simulations of membrane binding. We show that membrane binding by otoferlin involves C₂B-C₂G domains and repositions C₂F and C₂G domains. Disruption of Ca²⁺-binding sites of the C₂D domain in mice altered synaptic sound encoding and eliminated the Ca²⁺ cooperativity of IHC exocytosis, indicating that it requires the binding of several Ca²⁺-ions by otoferlin. Together, our findings elucidate molecular mechanisms underlying otoferlin-mediated SV docking and support the role of otoferlin as Ca²⁺ sensor of SV fusion in IHCs.