Calcium Regulates Molecular Interactions of Otoferlin with Soluble NSF Attachment Protein Receptor (SNARE) Proteins Required for Hair Cell Exocytosis

Abstract Mutations in otoferlin, a C2-domain-containing ferlin family protein, cause non-syndromic hearing loss in humans (DFNB9 deafness). Furthermore, transmitter secretion of cochlear inner hair cells is compromised in mice lacking otoferlin. In the present study we show that the C2F domain of otoferlin directly binds calcium (KD = 267 μM) with diminished binding in a pachanga (Asp1767Gly) C2F mouse mutation. Calcium is found to regulate differentially binding of otoferlin C2 domains to t-SNARE proteins and phospholipids. C2D-F domains interact with the syntaxin-1 t-SNARE motif, with a maximum binding within the range of 20-50 μM Ca2+. At 20 μM Ca2+, the dissociation rate is substantially lower, indicating increased binding (KD = ~10-9), compared to 0 μM Ca2+ (KD = ~10-8), suggesting a calcium-mediated stabilization of the C2 domain-t-SNARE complex. C2A and C2B interactions with t-SNAREs are insensitive to calcium. The C2F domain also directly binds the t-SNARE, SNAP-25, maximally at 100 μM and with reduction at 0 μM Ca2+, a pattern repeated for C2F domain interactions with PIP2. In contrast, C2F does not bind the v-SNARE protein synaptobrevin-1 (VAMP-1). Moreover, an antibody targeting otoferlin immunoprecipitates syntaxin-1 and SNAP-25 but not synaptobrevin-1. As opposed to an increase in binding with increased calcium, interactions between otoferlin C2F domain and intramolecular C2 domains occur in the absence of calcium, consistent with intra-C2 domain interactions forming a ″closed″ tertiary structure at low calcium that ″opens″ as calcium increases. These results suggest a direct role for otoferlin in exocytosis and modulation of calcium-dependent membrane fusion.