title: Otoferlin couples to clathrin-mediated endocytosis in mature cochlear inner hair cells.
abstract: The encoding of auditory information with indefatigable precision requires efficient resupply of vesicles at inner hair cell (IHC) ribbon synapses. Otoferlin, a transmembrane protein responsible for deafness in DFNB9 families, has been postulated to act as a calcium sensor for exocytosis as well as to be involved in rapid vesicle replenishment of IHCs. However, the molecular basis of vesicle recycling in IHCs is largely unknown. In the present study, we used high-resolution liquid chromatography coupled with mass spectrometry to copurify otoferlin interaction partners in the mammalian cochlea. We identified multiple subunits of the adaptor protein complex AP-2 (CLAP), an essential component of clathrin-mediated endocytosis, as binding partners of otoferlin in rats and mice. The interaction between otoferlin and AP-2 was confirmed by coimmunoprecipitation. We also found that AP-2 interacts with myosin VI, another otoferlin binding partner important for clathrin-mediated endocytosis (CME). The expression of AP-2 in IHCs was verified by reverse transcription PCR. Confocal microscopy experiments revealed that the expression of AP-2 and its colocalization with otoferlin is confined to mature IHCs. When CME was inhibited by blocking dynamin action, real-time changes in membrane capacitance showed impaired synaptic vesicle replenishment in mature but not immature IHCs. We suggest that an otoferlin-AP-2 interaction drives Ca(2+)- and stimulus-dependent compensating CME in mature IHCs.
authors.affiliation: Department of Pharmacology and Toxicology, Tübingen Hearing Research Centre, Molecular Physiology of Hearing, University of Tübingen, D-72076 Tübingen, Germany.
authors.forename: Susanne V
authors.initials: SV
authors.lastname: Duncker
authors.forename: Christoph
authors.initials: C
authors.lastname: Franz
authors.forename: Stephanie
authors.initials: S
authors.lastname: Kuhn
authors.forename: Uwe
authors.initials: U
authors.lastname: Schulte
authors.forename: Dario
authors.initials: D
authors.lastname: Campanelli
authors.forename: Niels
authors.initials: N
authors.lastname: Brandt
authors.forename: Bernhard
authors.initials: B
authors.lastname: Hirt
authors.forename: Bernd
authors.initials: B
authors.lastname: Fakler
authors.forename: Nikolaus
authors.initials: N
authors.lastname: Blin
authors.forename: Peter
authors.initials: P
authors.lastname: Ruth
authors.forename: Jutta
authors.initials: J
authors.lastname: Engel
authors.forename: Walter
authors.initials: W
authors.lastname: Marcotti
authors.forename: Ulrike
authors.initials: U
authors.lastname: Zimmermann
authors.forename: Marlies
authors.initials: M
authors.lastname: Knipper
mesh_terms: D033962:Adaptor Protein Complex 2 / Q000502:physiology; D000818:Animals; D002462:Cell Membrane / Q000502:physiology; D002966:Clathrin / Q000502:physiology*; D003051:Cochlea / Q000166:cytology / Q000502:physiology*; D055724:Electrophysiological Phenomena; D004705:Endocytosis / Q000502:physiology*; D006199:Hair Cells, Auditory, Inner / Q000502:physiology*; D007150:Immunohistochemistry; D047468:Immunoprecipitation; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000502:physiology*; D051379:Mice; D018613:Microscopy, Confocal; D018995:Myosin Heavy Chains / Q000502:physiology; D011485:Protein Binding; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D051381:Rats; D017208:Rats, Wistar; D060888:Real-Time Polymerase Chain Reaction; D013569:Synapses / Q000502:physiology
chemical_list: D033962:Adaptor Protein Complex 2; D002966:Clathrin; D008565:Membrane Proteins; C533993:Otof protein, rat; D012333:RNA, Messenger; C089785:myosin VI; D018995:Myosin Heavy Chains