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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 Ca2+ influx and exocytosis, which we attribute to a decreased abundance of presynaptic Ca2+ 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 Ca2+ influx and promote SV recycling in IHCs, likely via coupling exocytosis to endocytosis, and contributing to membrane retrieval and SV reformation.

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.