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 2ϩ -and stimulus-dependent compensating CME in mature IHCs.

Otoferlin Deficiency in Zebrafish Results in Defects in Balance and Hearing: Rescue of the Balance and Hearing Phenotype with Full-Length and Truncated Forms of Mouse Otoferlin

“…3G). This subcellular distribution is similar to observations made on mouse hair cells (20,23,37). Probing 120-h wild-type zebrafish with the FM1-43 dye (Fig.…”

“…Otoferlin is a six-C2 domain transmembrane protein expressed in inner, outer, and vestibular hair cells, as well as restricted regions of the brain (13-16). In humans, missense mutations in otoferlin have been linked to hearing loss (17,18), and biochemical studies have determined that otoferlin binds calcium and lipids (12,19), as well as membrane trafficking proteins (12,(20)(21)(22)(23). Further, in vitro assays have demonstrated that otoferlin accelerates SNARE-mediated membrane fusion (12).…”

Audibility, speech perception and processing of temporal cues in ribbon synaptic disorders due to OTOF mutations

“…Otoferlin has recently been found to interact with AP-2 and myosin VI, two proteins involved in clathrin-mediated endocytosis (Duncker et al, 2013), which is essential to synaptic vesicle replenishment. On these grounds a further role for otoferlin has been suggested: it could be involved in coupling exocytosis and endocytosis in IHCs, thus providing the basis for sustained release at the ribbon synapse.…”

Auditory neuropathy — neural and synaptic mechanisms

“…4). Otoferlin could prime synaptic vesicles for fusion 19 , potentially via promoting the for mation of short tethers between synaptic vesicles and the membrane of the active zone, as suggested by elec tron tomography 29 , and/or it could facilitate the clear ance of previously exocytosed material from the release site via interaction with endocytic proteins 18,63 . Missense mutations might affect these processes by lower ing the otoferlin levels or by impairing its interactions with other proteins.…”