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. Here, we report that the endocytic adaptor protein 2l (AP-2l) is required for release site replenishment and hearing. We show that hair cell-specific disruption of AP-2l 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. Sound-driven postsynaptic spiking was reduced in a use-dependent manner, and the altered interspike interval statistics suggested a slowed reloading of release sites. Sustained strong stimulation led to accumulation of endosomelike vacuoles, fewer clathrin-coated endocytic intermediates, and vesicle depletion of the membrane-distal synaptic ribbon in AP-2l-deficient IHCs, indicating a further role of AP-2l in clathrin-dependent vesicle reformation on a timescale of many seconds. Finally, we show that AP-2 sorts its IHC-cargo otoferlin. 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.

Ca 2+ -binding protein 2 (CaBP2) inhibits the inactivation of heterologously expressed voltage-gated Ca 2+ channels of type 1.3 (Ca V 1.3) and is defective in human autosomal-recessive deafness 93 (DFNB93). Here, we report a newly identified mutation in CABP2 that causes a moderate hearing impairment likely via nonsense-mediated decay of CABP2-mRNA. To study the mechanism of hearing impairment resulting from CABP2 loss of function, we disrupted Cabp2 in mice (Cabp2 LacZ/LacZ ). CaBP2 was expressed by cochlear hair cells, preferentially in inner hair cells (IHCs), and was lacking from the postsynaptic spiral ganglion neurons (SGNs). Cabp2 LacZ/LacZ mice displayed intact cochlear amplification but impaired auditory brainstem responses. Patch-clamp recordings from Cabp2 LacZ/LacZ IHCs revealed enhanced Ca 2+ -channel inactivation. The voltage dependence of activation and the number of Ca 2+ channels appeared normal in Cabp2 LacZ/LacZ mice, as were ribbon synapse counts. Recordings from single SGNs showed reduced spontaneous and sound-evoked firing rates. We propose that CaBP2 inhibits Ca V 1.3 Ca 2+ -channel inactivation, and thus sustains the availability of Ca V 1.3 Ca 2+ channels for synaptic sound encoding. Therefore, we conclude that human deafness DFNB93 is an auditory synaptopathy.H earing relies on faithful transmission of information at ribbon synapses between inner hair cells (IHCs) and spiral ganglion neurons (SGNs; recently reviewed in refs. 1, 2). Ca 2+ channels at the IHC presynaptic active zone are key signaling elements because they couple the sound-evoked IHC receptor potential to the release of glutamate. IHC Ca 2+ -channel complexes are known to contain Ca V 1.3 α1 subunit (Cav1.3α1) (3-5), betasubunit 2 (Ca V β2) (6), and alpha2-delta subunit 2 (α2δ2) (7) to activate at around −60 mV (8-10), and are partially activated already at the IHC resting potential in vivo [thought to be between −55 and −45 mV (11, 12)], thereby mediating "spontaneous" glutamate release during silence (13).Compared with Ca V 1.3 channels studied in heterologous expression systems, Ca V 1.3 channels in IHCs show little inactivation, which has been attributed to inhibition of calmodulin-mediated Ca 2+ -dependent inactivation (CDI) (14-17) by Ca 2+ -binding proteins (CaBPs) (18,19) and/or the interaction of the distal and proximal regulatory domains of the Ca V 1.3α1 C terminus (20)(21)(22). This "noninactivating" phenotype of IHC Ca V 1.3 enables reliable excitation-secretion coupling during ongoing stimulation (23-25). In fact, postsynaptic spike rate adaptation during ongoing sound stimulation is thought to reflect primarily presynaptic vesicle pool depletion, with minor contributions of Ca V 1.3 inactivation or AMPA-receptor desensitization (23-26). CaBPs are calmodulin-like proteins that use three functional out of four helix-loop-helix domains (EF-hand) for Ca 2+ binding (27). They are thought to function primarily as signaling proteins (28) and differentially modulate calmodulin effectors (29,30). In addition, CaBPs m...

Otoferlin is essential for fast Ca2+-triggered transmitter release from auditory inner hair cells (IHCs), playing key roles in synaptic vesicle release, replenishment and retrieval. Dysfunction of otoferlin results in profound prelingual deafness. Despite its crucial role in cochlear synaptic processes, mechanisms regulating otoferlin activity have not been studied to date. Here, we identified Ca2+/calmodulin-dependent serine/threonine kinase II delta (CaMKIIδ) as an otoferlin binding partner by pull-downs from chicken utricles and reassured interaction by a co-immunoprecipitation with heterologously expressed proteins in HEK cells. We confirmed the expression of CaMKIIδ in rodent IHCs by immunohistochemistry and real-time PCR. A proximity ligation assay indicates close proximity of the two proteins in rat IHCs, suggesting that otoferlin and CaMKIIδ also interact in mammalian IHCs. In vitro phosphorylation of otoferlin by CaMKIIδ revealed ten phosphorylation sites, five of which are located within C2-domains. Exchange of serines/threonines at phosphorylated sites into phosphomimetic aspartates reduces the Ca2+ affinity of the recombinant C2F domain 10-fold, and increases the Ca2+ affinity of the C2C domain. Concordantly, we show that phosphorylation of otoferlin and/or its interaction partners are enhanced upon hair cell depolarization and blocked by pharmacological CaMKII inhibition. We therefore propose that otoferlin activity is regulated by CaMKIIδ in IHCs.