Cryo-EM structures of otoferlin reveal a compact ring-like architecture that binds membranes through a multi-domain Ca2+-dependent interface involving nine coordinated Ca2+ ions, and structure-guided mutagenesis of Ca2+-binding sites in mice demonstrates that otoferlin functions as the Ca2+-sensor of synaptic vesicle fusion in inner hair cells by showing that progressive disruption of C2D domain Ca2+-binding eliminates the Ca2+-cooperativity of exocytosis while reducing release probability without impairing vesicle replenishment.

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-C2-domain protein otoferlin which 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 Ca2+-bound otoferlin and employed molecular dynamics simulations of membrane binding. We show that membrane binding involves C2B-C2G-domains and repositions C2F- and C2G-domains. Progressive disruption of Ca2+-binding by the C2D-domain in mice increasingly altered synaptic sound encoding and eliminated the Ca2+-cooperativity of SV-exocytosis, indicating that this Ca2+-cooperativity reflects binding of several Ca2+-ions to otoferlin. Together, our findings elucidate molecular mechanisms underlying otoferlin-mediated SV-docking and support a role of otoferlin as Ca2+-sensor of SV-fusion in IHCs.