Hearing relies on rapid, temporally precise, and sustained neurotransmitter release at the ribbon synapses of sensory cells, the inner hair cells (IHCs). This process requires otoferlin, a six C2-domain, Ca2+-binding transmembrane protein of synaptic vesicles. To decipher the role of otoferlin in the synaptic vesicle cycle, we produced knock-in mice (Otof Ala515,Ala517/Ala515,Ala517) with lower Ca2+-binding affinity of the C2C domain. The IHC ribbon synapse structure, synaptic Ca2+ currents, and otoferlin distribution were unaffected in these mutant mice, but auditory brainstem response wave-I amplitude was reduced. Lower Ca2+ sensitivity and delay of the fast and sustained components of synaptic exocytosis were revealed by membrane capacitance measurement upon modulations of intracellular Ca2+ concentration, by varying Ca2+ influx through voltage-gated Ca2+-channels or Ca2+ uncaging. Otoferlin thus functions as a Ca2+ sensor, setting the rates of primed vesicle fusion with the presynaptic plasma membrane and synaptic vesicle pool replenishment in the IHC active zone.

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Transmitter release at auditory inner hair cell (IHC) ribbon synapses involves exocytosis of glutamatergic vesicles during voltage activation of L-type Ca v 1.3 calcium channels. At these synapses, the fast and indefatigable release of synaptic vesicles by IHCs is controlled by otoferlin, a six-C2-domain (C2-ABCDEF) protein that functions as a high-affinity Ca 2ϩ sensor. The molecular events by which each otoferlin C2 domain contributes to the regulation of the synaptic vesicle cycle in IHCs are still incompletely understood. Here, we investigate their role using a cochlear viral cDNA transfer approach in vivo, where IHCs of mouse lacking otoferlin (Otof Ϫ/Ϫ mice of both sexes) were virally transduced with cDNAs of various mini-otoferlins. Using patch-clamp recordings and membrane capacitance measurements, we show that the viral transfer of mini-otoferlin containing C2-ACEF, C2-EF, or C2-DEF partially restores the fast exocytotic component in Otof Ϫ/Ϫ mouse IHCs. The restoration was much less efficient with C2-ACDF, underlining the importance of the C2-EF domain. None of the mini-otoferlins tested restored the sustained component of vesicle release, explaining the absence of hearing recovery. The restoration of the fast exocytotic component in the transduced Otof Ϫ/Ϫ IHCs was also associated with a recovery of Ca 2ϩ currents with normal amplitude and fast time inactivation, confirming that the C-terminal C2 domains of otoferlin are essential for normal gating of Ca v 1.3 channels. Finally, the reintroduction of the mini-otoferlins C2-EF, C2-DEF, or C2-ACEF allowed us to uncover and characterize for the first time a dynamin-dependent ultrafast endocytosis in IHCs.