Probing the role of the C2F domain of otoferlin.

Afferent synapses of cochlear inner hair cells (IHCs) employ a unique molecular machinery. Otoferlin is a key player in this machinery, and its genetic defects cause human auditory synaptopathy. We employed site-directed mutagenesis in mice to investigate the role of Ca²⁺ binding to the C₂F domain of otoferlin. Substituting two aspartate residues of the C₂F top loops, which are thought to coordinate Ca²⁺- ions, by alanines (Otof^D1841/1842A) abolished Ca²⁺-influx-triggered IHC exocytosis and synchronous signaling in the auditory pathway despite substantial expression (~60%) of the mutant otoferlin in the basolateral IHC pole. Ca²⁺ influx of IHCs and their resting membrane capacitance, reflecting IHC size, as well as the number of IHC synapses were maintained. The mutant otoferlin showed a strong apexto-base abundance gradient in IHCs, suggesting impaired protein targeting. Our results indicate a role of the C₂F domain in otoferlin targeting and of Ca²⁺ binding by the C₂F domain for IHC exocytosis and hearing. [ABSTRACT FROM AUTHOR]