Otoferlin acts as a Ca2+ sensor for vesicle fusion and vesicle pool replenishment at auditory hair cell ribbon synapses

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 (<i>Otof</i>^{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.