PKCα-dependent interaction of otoferlin and calbindin: evidence for regulation of endocytosis in inner hair cells

Otoferlin is essential for the fast and indefatigable release of synaptic vesicles at auditory inner hair cell (IHC) ribbon synapses, being involved in exocytic, endocytic and regenerative steps of the synaptic vesicle cycle. Serving diverse functions at this highly dynamic synapse implies that this multi-C2 domain protein is precisely regulated. Here we found protein kinase C (PKC) and otoferlin to colocalize in endocytic recycling compartments upon IHC depolarization and to interact in an activity-dependent manner. In vitro assays confirmed that PKC can phosphorylate otoferlin at five serine residues, which correlates with increased serine phosphorylation in <40 nm proximity to otoferlin in murine IHCs that can be fully blocked by combining PKC and CaMKII inhibitors. Moreover, otoferlin interacts with calbindin-D28k in stimulated IHCs, which was precluded when PKC was inhibited. Similarly, the activity-dependent increase in otoferlin-myosin VI interaction depends on PKC activation. We propose that upon strong hair cell depolarization, PKC phosphorylates otoferlin, thereby enabling it to interact with calbindin-D28k and myosin VI, building a Ca2+-dependent signaling complex that possibly regulates different modes of endocytosis.