A Novel Single Molecule Assay Reveals the Deafness‐Associated Protein Otoferlin as a Multivalent Calcium‐Sensitive Scaffold Linking SNAREs and Calcium Channels

Abstract

Sensory hair cells rely on otoferlin as the calcium sensor for exocytosis and encoding of sound preferentially over the neuronal calcium sensor synaptotagmin. Although it is established that synaptotagmin cannot rescue the otoferlin KO phenotype, the large size and low solubility of otoferlin have prohibited direct biochemical comparisons that could establish functional differences between these two proteins. To address this challenge, we have developed a singlemolecule colocalization binding titration assay (smCoBRA) that can quantitatively characterize full-length otoferlin from mammalian cell lysate. Using smCoBRA, we found that, although both otoferlin and synaptotagmin bind membrane fusion SNARE proteins, only otoferlin interacts with the L-type calcium channel Cav1.3, showing a significant difference between the synaptic proteins. Furthermore, otoferlin was found capable of interacting with multiple SNARE and Cav1.3 proteins simultaneously, forming a heterooligomer complex. We also found that a deafness-causing missense mutation in otoferlin attenuates binding between otoferlin and Cav1.3, suggesting that deficiencies in this interaction may form the basis for otoferlin-related hearing loss. Based on our results, we propose a model in which otoferlin acts as a calcium-sensitive scaffolding protein, localizing SNARE proteins proximal to the calcium channel so as to synchronize calcium influx with membrane fusion. Our findings also provide a molecular-level explanation for the observation that synaptotagmin and otoferlin are not functionally redundant. This study also validates Q:9 a generally applicable methodology for quantitatively characterizing large, multivalent membrane proteins. This presentation is based on our recent publication (Hams et al. Proc Natl Acad Sci 2017 114:8023-8028). Support or Funding Information NIH NIDCD Grant 1R01DC014588 Schematic of sensory hair cell and neuronal presynapses. (A) Synapticribbons within the sensory hair cells of the cochlea position synaptic vesiclesproximal to the presynaptic membrane. Otoferlin resides on synaptic vesicles, whereas Cav1.3 localizes to the presynapse. (B) The synaptic vesicles of neurons harbor synaptotagmin I/II and Cav2.2). (C) Diagram of otoferlin depicting six C2 domains, labeled C2A–C2F, and the transmembrane domain (TMD). This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.