The transmembrane recognition complex (TRC40) pathway mediates the insertion of tail‐anchored (TA) proteins into membranes. Here, we demonstrate that otoferlin, a TA protein essential for hair cell exocytosis, is inserted into the endoplasmic reticulum (ER) via the TRC40 pathway. We mutated the TRC40 receptor tryptophan‐rich basic protein (Wrb) in hair cells of zebrafish and mice and studied the impact of defective TA protein insertion. Wrb disruption reduced otoferlin levels in hair cells and impaired hearing, which could be restored in zebrafish by transgenic Wrb rescue and otoferlin overexpression. Wrb‐deficient mouse inner hair cells (IHCs) displayed normal numbers of afferent synapses, Ca2+ channels, and membrane‐proximal vesicles, but contained fewer ribbon‐associated vesicles. Patch‐clamp of IHCs revealed impaired synaptic vesicle replenishment. In vivo recordings from postsynaptic spiral ganglion neurons showed a use‐dependent reduction in sound‐evoked spiking, corroborating the notion of impaired IHC vesicle replenishment. A human mutation affecting the transmembrane domain of otoferlin impaired its ER targeting and caused an auditory synaptopathy. We conclude that the TRC40 pathway is critical for hearing and propose that otoferlin is an essential substrate of this pathway in hair cells.
Otoferlin, a tail‐anchored protein of sensory hair cells, uses the transmembrane recognition complex (TRC40) pathway for ER membrane insertion. Disruption of the TRC40 receptor Wrb in hair cells impaired vesicle replenishment and hearing, most likely by reducing otoferlin levels.
Hearing requires membrane insertion of tail‐anchored (TA) proteins by the TRC40 pathway in hair cells.
The TA protein otoferlin, essential for hair cell exocytosis and defective in human deafness, utilizes the TRC40 pathway.
Deletion of TRC40 receptor WRB reduces otoferlin levels and impairs synaptic structure and function.
Reduced vesicle replenishment in WRB‐deficient hair cells impairs sound encoding.
- Received November 30, 2015.
- Revision received May 29, 2016.
- Accepted June 10, 2016.
- © 2016 The Authors
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