Probing the role of the C2F domain of otoferlin

Abstract

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 Ca 2+ binding to the C 2 F domain of otoferlin. Substituting two aspartate residues of the C 2 F top loops, which are thought to coordinate Ca 2+ -ions, by alanines ( Otof D1841/1842A ) abolished Ca 2+ -influx-triggered IHC exocytosis and synchronous signaling in the auditory pathway despite substantial expression (~60%) of the mutant otoferlin in the basolateral IHC pole. Ca 2+ 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 apex-to-base abundance gradient in IHCs, suggesting impaired protein targeting. Our results indicate a role of the C 2 F domain in otoferlin targeting and of Ca 2+ binding by the C 2 F domain for IHC exocytosis and hearing.