Endophilin‐A regulates presynaptic Ca2+ influx and synaptic vesicle recycling in auditory hair cells

Ribbon synapses of cochlear inner hair cells (IHCs) operate with high rates of neurotransmission; yet, the molecular regulation of synaptic vesicle (SV) recycling at these synapses remains poorly understood. Here, we studied the role of endophilins‐A1‐3, endocytic adaptors with curvature‐sensing and curvature‐generating properties, in mouse IHCs. Single‐cell RT–PCR indicated the expression of endophilins‐A1‐3 in IHCs, and immunoblotting confirmed the presence of endophilin‐A1 and endophilin‐A2 in the cochlea. Patch‐clamp recordings from endophilin‐A‐deficient IHCs revealed a reduction of Ca 2+ influx and exocytosis, which we attribute to a decreased abundance of presynaptic Ca 2+ channels and impaired SV replenishment. Slow endocytic membrane retrieval, thought to reflect clathrin‐mediated endocytosis, was impaired. Otoferlin, essential for IHC exocytosis, co‐immunoprecipitated with purified endophilin‐A1 protein, suggestive of a molecular interaction that might aid exocytosis–endocytosis coupling. Electron microscopy revealed lower SV numbers, but an increased occurrence of coated structures and endosome‐like vacuoles at IHC active zones. In summary, endophilins regulate Ca 2+ influx and promote SV recycling in IHCs, likely via coupling exocytosis to endocytosis, and contributing to membrane retrieval and SV reformation. Synopsis Using a multidisciplinary approach, we show that endophilin‐A positively regulates presynaptic Ca 2+ influx and interacts with otoferlin. Moreover, endophilin‐A supports vesicle endocytosis and clathrin‐dependent vesicle reformation at ribbon synapses of murine inner hair cells. Absence of endophilin‐A leads to reduced presynaptic Ca 2+ channel cluster size and attenuated Ca 2+ influx at inner hair cell ribbon synapses. Otoferlin physically interacts with endophilin; loss of endophilin‐A1 and ‐A3 leads to a reduction in otoferlin levels of ˜25%. The number of cytosolic and ribbon‐associated SVs is decreased in mutants missing several endophilin‐A genes; consequently, IHC sustained exocytosis was found to be reduced. Absence of endophilin‐A leads to accumulations of endosome‐like vacuoles and clathrin‐coated organelles; the severity of the phenotype depends on the number of missing endophilin alleles. Graphical Abstract Endophilins regulate Ca 2+ channel abundance, Ca 2+ influx and synaptic vesicle recycling in inner hair cells by coupling exocytosis to endocytosis, contributing to membrane retrieval and synaptic vesicle reformati