Abstract
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-C(2) domain protein otoferlin that 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 otoferlin and employed molecular dynamics simulations of membrane binding. We show that membrane binding by otoferlin involves C(2)B-C(2)G domains and repositions C(2)F and C(2)G domains. Disruption of Ca(2+)-binding sites of the C(2)D domain in mice altered synaptic sound encoding and eliminated the Ca(2+) cooperativity of IHC exocytosis, indicating that it requires the binding of several Ca(2+)-ions by otoferlin. Together, our findings elucidate molecular mechanisms underlying otoferlin-mediated SV docking and support the role of otoferlin as Ca(2+) sensor of SV fusion in IHCs.