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

Our sense of hearing relies upon speedy synaptic transmission of sound information from cochlear inner hair cells (IHCs) to spiral ganglion neurons (SGNs). To accomplish this, IHCs employ a sophisticated presynaptic machinery including the multi-C ₂ -domain protein otoferlin which is affected by human deafness mutations. Otoferlin is essential for IHC-exocytosis but how it binds Ca ²⁺ and the target membrane to serve synaptic vesicle (SV) tethering, docking and fusion remained unclear. Here, we obtained cryo-electron-microscopy structures of Ca ²⁺ -bound otoferlin and employed molecular dynamics simulations of membrane binding. We show that membrane binding involves C ₂ B-C ₂ G-domains and repositions C ₂ F- and C ₂ G-domains. Progressive disruption of Ca ²⁺ -binding by the C ₂ D-domain in mice increasingly altered synaptic sound encoding and eliminated the Ca ²⁺ -cooperativity of SV-exocytosis, indicating that this Ca ²⁺ -cooperativity reflects binding of several Ca ²⁺ -ions to otoferlin. Together, our findings elucidate molecular mechanisms underlying otoferlin-mediated SV-docking and support a role of otoferlin as Ca ²⁺ -sensor of SV-fusion in IHCs.