Activity-Dependent Phosphorylation by CaMKIIδ Alters the Ca2+ Affinity of the Multi-C2-Domain Protein Otoferlin.

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