Probing the function of Ca2+-binding C2 domains of otoferlin, a synaptic protein of sensory hair cells

by Han Chen
Doctoral thesis
Date of Examination:2023-12-15
Date of issue:2024-11-05
Advisor:Prof. Dr. Tobias Moser
Referee:Prof. Dr. Silvio Rizzoli
Referee:Prof. Dr. Erwin Neher
Referee:Prof. Dr. Nils Brose
Referee:Prof. Dr. Thomas Dresbach
Referee:Prof. Dr. Martin Göpfert
Referee:Dr. Eri Sakata
Persistent Address: http://dx.doi.org/10.53846/goediss-10840

 

 

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Abstract

English

The ribbon synapses in auditory inner hair cells (IHCs) are specialized to achieve indefatigable sound encoding at rates of hundreds of Hertz and with sub-millisecond temporal precision. While conventional synapses employ the SNARE complex and its regulatory proteins such as synaptotagmins, Munc13 and Munc18, IHCs do not seem to express most of these proteins. IHC ribbon synapses employ an unconventional molecular machinery to mediate Ca2+-triggered exocytosis. Otoferlin, specifically and strongly expressed in IHCs, is a key player of this machinery and essential for exocytosis in IHCs. In this study, we aimed to test the hypothesis that otoferlin is the Ca2+ sensor of synaptic vesicle fusion in IHCs. We approached this question by characterizing the functional contribution of distinct otoferlin C2 domains. In particular, we investigated the function of the C2D, C2E and C2F domains by employing site-directed mutagenesis in mice. Substituting two predicted Ca2+-binding aspartates of the C2F domain resulted in abnormal otoferlin subcellular distribution and abolished exocytosis. Substitution three potential Ca2+ binding aspartates of the C2E domain led to disrupted exocytosis and hearing despite substantial expression and normal subcellular distribution of otoferlin. Exocytosis was partially rescued by applying extremely high Ca2+ to the IHCs, which is in line with a lower Ca2+ binding affinity of the mutant otoferlin. All these findings support the Ca2+ sensor hypothesis for otoferlin. The OTOFIT (I1573T) mutation is a mouse model of a human missense mutation affecting the C2E domain. In this case, we found greatly reduced otoferlin level (~20%). This suggested that the C2E domain is critical for the folding and stability of otoferlin, and it is in line with the analysis of otoferlin’s tertiary structure. Lastly, we substituted potential Ca2+-binding aspartates of the C2D domain. According to the AlphaFold2 structure and also hinted by the fact that there are fewer pathogenic mutations found on the C2D domain, we expected a milder phenotype for C2D mutants. This way we could take advantage for investigating more details of the function of otoferlin in the synaptic vesicle cycle. Substituting Ca2+-binding aspartates of the C2D domain led to otoferlin expression indistinguishable to wild-type, but showed reduced exocytosis even for short term stimulations (< 20ms) which suggests altered synaptic vesicle fusion. In summary, this work indicates critical roles of Ca2+ binding to the C2D, C2E and C2F domains for otoferlin function, IHC exocytosis and hearing. We proposed that the C2F domain is important for subcellular targeting of otoferlin, the C2E domain is essential for the stability of the protein, and C2D, C2E and C2F domains contribute to Ca2+ sensing for IHC exocytosis.
Keywords: hearing; deafness; otoferlin; inner hair cell; cochlea; exocytosis; ribbon synapse
 
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