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Otoferlin is a calcium sensor that directly regulates SNARE-mediated membrane fusion
Colin P Johnson1, Edwin R Chapman
1Howard Hughes Medical Institute, University of Wisconsin, Madison, WI 53706, USA.
Abstract:
Otoferlin is a large multi-C2 domain protein proposed to act as a calcium sensor that regulates synaptic vesicle exocytosis in cochlear hair cells. Although mutations in otoferlin have been associated with deafness, its contribution to neurotransmitter release is unresolved. Using recombinant proteins, we demonstrate that five of the six C2 domains of otoferlin sense calcium with apparent dissociation constants that ranged from 13-25 µM; in the presence of membranes, these apparent affinities increase by up to sevenfold. Using a reconstituted membrane fusion assay, we found that five of the six C2 domains of otoferlin stimulate membrane fusion in a calcium-dependent manner. We also demonstrate that a calcium binding-deficient form of the C2C domain is incapable of stimulating membrane fusion, further underscoring the importance of calcium for the protein's function. These results demonstrate for the first time that otoferlin is a calcium sensor that can directly regulate soluble N-ethyl-maleimide sensitive fusion protein attachment protein receptor-mediated membrane fusion reactions.
Insights
Otoferlin acts as a calcium sensor in hearing cells, directly regulating membrane fusion essential for neurotransmitter release. This finding clarifies otoferlin
Area of Science:
- Molecular Biology
- Neuroscience
- Auditory Science
Background:
- Otoferlin, a protein with multiple C2 domains, is implicated in synaptic vesicle exocytosis in cochlear hair cells.
- Mutations in otoferlin are linked to deafness, but its precise role in neurotransmitter release remains unclear.
Purpose of the Study:
- To elucidate the function of otoferlin as a calcium sensor in regulating neurotransmitter release.
- To investigate the calcium-binding properties and membrane fusion capabilities of otoferlin's C2 domains.
Main Methods:
- Utilized recombinant proteins to study otoferlin's C2 domains.
- Employed a reconstituted membrane fusion assay to assess otoferlin's function.
- Investigated calcium-dependent membrane fusion and the role of specific C2 domains.
Main Results:
- Five of otoferlin's six C2 domains sense calcium, with affinities enhanced by membranes.
- These C2 domains stimulate membrane fusion in a calcium-dependent manner.
- A calcium-binding deficient mutant of the C2C domain failed to stimulate membrane fusion.
Conclusions:
- Otoferlin functions as a direct calcium sensor.
- It regulates membrane fusion reactions, specifically soluble N-ethyl-maleimide sensitive fusion protein attachment receptor (SNARE)-mediated fusion.
- This provides a molecular mechanism for otoferlin's role in auditory neurotransmission.
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