Abstract
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.
Highlights
Otoferlin is a 230 kDa, tail-anchored membrane protein, containing at least six C2 domains implicated in Ca2+, phospholipid, and protein binding (Yasunaga et al, 1999; Johnson and Chapman, 2010; Pangršicet al., 2012)
In order to identify interaction partners of otoferlin, we used embryonic day 18 (E18) vestibular maculae of the chicken utricle, each containing more than 20000 hair cells for affinity purification of otoferlin
In mouse organs of Corti at P14 we found hardly any immunolabelling for CaMKIIα, CaMKIIβ or CaMKIIγ within the cytoplasm of inner hair cell (IHC), which were co-labeled for otoferlin (Figures 1A–C)
Summary
Otoferlin is a 230 kDa, tail-anchored membrane protein, containing at least six C2 domains implicated in Ca2+, phospholipid, and protein binding (Yasunaga et al, 1999; Johnson and Chapman, 2010; Pangršicet al., 2012). Several protein interaction partners of otoferlin have been reported including myosin VI, Rab8b, SNARE proteins, Cav1.3 Ca2+ channel, Ergic and AP-2 (Roux et al, 2006; Heidrych et al, 2008, 2009; Ramakrishnan et al, 2009; Roux et al, 2009; Zak et al, 2012; Duncker et al, 2013; Jung et al, 2015).
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