| Autor: |
Meese S; Department of Molecular Structural Biology, Institute for Microbiology and Genetics, and Collaborative Research Center 889, University of Göttingen, Göttingen, Germany.; Göttingen Graduate School for Neurosciences, Biophysics, and Molecular Biosciences, Göttingen, Germany., Cepeda AP; Göttingen Graduate School for Neurosciences, Biophysics, and Molecular Biosciences, Göttingen, Germany.; Molecular Biology of Cochlear Neurotransmission Group, Department of Otorhinolaryngology, University Medical Center Göttingen, and Collaborative Research Center 889, University of Göttingen, Göttingen, Germany., Gahlen F; Department of Otorhinolaryngology, Head and Neck Surgery, Ruhr-University Bochum, Bochum, Germany., Adams CM; Stanford University Mass Spectrometry, Stanford University, Stanford, CA, United States., Ficner R; Department of Molecular Structural Biology, Institute for Microbiology and Genetics, and Collaborative Research Center 889, University of Göttingen, Göttingen, Germany., Ricci AJ; Department of Otolaryngology, Head and Neck Surgery, Stanford School of Medicine, Stanford, CA, United States., Heller S; Department of Otolaryngology, Head and Neck Surgery, Stanford School of Medicine, Stanford, CA, United States., Reisinger E; Molecular Biology of Cochlear Neurotransmission Group, Department of Otorhinolaryngology, University Medical Center Göttingen, and Collaborative Research Center 889, University of Göttingen, Göttingen, Germany., Herget M; Department of Otolaryngology, Head and Neck Surgery, Stanford School of Medicine, Stanford, CA, United States. |
| Abstrakt: |
Otoferlin is essential for fast Ca 2+ -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 Ca 2+ /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 C 2 -domains. Exchange of serines/threonines at phosphorylated sites into phosphomimetic aspartates reduces the Ca 2+ affinity of the recombinant C 2 F domain 10-fold, and increases the Ca 2+ affinity of the C 2 C 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. |
