![]() Dissociation of vesicular proteins from the release site and/or diffusion of newly exocytosed material to the perisynaptic zone of endocytosis might be rate‐limiting the rapid release site cycle in IHCs. However, a role of otoferlin in pre‐fusion priming could so far not be distinguished from a post‐fusion function in clearing previously exocytosed membrane from release sites (Pangršič et al, 2010 Pangršič et al, 2012 Duncker et al, 2013). Rapid vesicle turnover requires sufficient amounts of the hair cell C 2‐domain protein otoferlin (Roux et al, 2006 Pangršič et al, 2010) that when defective causes human hearing impairment (Yasunaga et al, 1999 Varga et al, 2006). Indeed, sustained exocytosis of up to 70 Hz from each of the ~10–15 release sites of the AZ composing the readily releasable pool (RRP) was reported (Pangršič et al, 2010) requiring rapid reloading. Such vivid firing requires the AZ to indefatigably release vesicles at even higher rates, because some release events fail to trigger a spike due to neural refractoriness. ![]() Even during continued stimulation, each SGN can fire at hundreds of Hz in response to release from a single IHC AZ (Matthews & Fuchs, 2010 Pangršič et al, 2012 Safieddine et al, 2012). ![]() Hearing relies on sound encoding at ribbon synapses between IHCs and spiral ganglion neurons (SGNs). We propose that binding of AP‐2 to otoferlin facilitates replenishment of release sites, for example, via speeding AZ clearance of exocytosed material, in addition to a role of AP‐2 in synaptic vesicle reformation. Finally, we show that AP‐2 sorts its IHC‐cargo otoferlin. Sustained strong stimulation led to accumulation of endosome‐like vacuoles, fewer clathrin‐coated endocytic intermediates, and vesicle depletion of the membrane‐distal synaptic ribbon in AP‐2μ‐deficient IHCs, indicating a further role of AP‐2μ in clathrin‐dependent vesicle reformation on a timescale of many seconds. Sound‐driven postsynaptic spiking was reduced in a use‐dependent manner, and the altered interspike interval statistics suggested a slowed reloading of release sites. We show that hair cell‐specific disruption of AP‐2μ slows IHC exocytosis immediately after fusion of the readily releasable pool of vesicles, despite normal abundance of membrane‐proximal vesicles and intact endocytic membrane retrieval. ![]() ![]() Here, we report that the endocytic adaptor protein 2μ ( AP‐2μ) is required for release site replenishment and hearing. Active zones ( AZs) of inner hair cells ( IHCs) indefatigably release hundreds of vesicles per second, requiring each release site to reload vesicles at tens per second. ![]()
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