title: Unconventional molecular regulation of synaptic vesicle replenishment in cochlear inner hair cells.
abstract: Ribbon synapses of cochlear inner hair cells (IHCs) employ efficient vesicle replenishment to indefatigably encode sound. In neurons, neuroendocrine and immune cells, vesicle replenishment depends on proteins of the mammalian uncoordinated 13 (Munc13, also known as Unc13) and Ca(2+)-dependent activator proteins for secretion (CAPS) families, which prime vesicles for exocytosis. Here, we tested whether Munc13 and CAPS proteins also regulate exocytosis in mouse IHCs by combining immunohistochemistry with auditory systems physiology and IHC patch-clamp recordings of exocytosis in mice lacking Munc13 and CAPS isoforms. Surprisingly, we did not detect Munc13 or CAPS proteins at IHC presynaptic active zones and found normal IHC exocytosis as well as auditory brainstem responses (ABRs) in Munc13 and CAPS deletion mutants. Instead, we show that otoferlin, a C2-domain protein that is crucial for vesicular fusion and replenishment in IHCs, clusters at the plasma membrane of the presynaptic active zone. Electron tomography of otoferlin-deficient IHC synapses revealed a reduction of short tethers holding vesicles at the active zone, which might be a structural correlate of impaired vesicle priming in otoferlin-deficient IHCs. We conclude that IHCs use an unconventional priming machinery that involves otoferlin.
authors.affiliation: Institute for Auditory Neuroscience and InnerEarLab, Department of Otolaryngology, University Medical Center Göttingen, 37099 Göttingen, Germany christian.vogl@med.uni-goettingen.de tmoser@gwdg.de carolin.wichmann@med.uni-goettingen.de.
authors.forename: Christian
authors.initials: C
authors.lastname: Vogl
authors.affiliation: Department of Molecular Neurobiology, Max Planck Institute of Experimental Medicine, 37075 Göttingen, Germany.
authors.forename: Benjamin H
authors.initials: BH
authors.lastname: Cooper
authors.affiliation: Institute for Auditory Neuroscience and InnerEarLab, Department of Otolaryngology, University Medical Center Göttingen, 37099 Göttingen, Germany.
authors.forename: Jakob
authors.initials: J
authors.lastname: Neef
authors.affiliation: Department of Molecular Neurobiology, Max Planck Institute of Experimental Medicine, 37075 Göttingen, Germany.
authors.forename: Sonja M
authors.initials: SM
authors.lastname: Wojcik
authors.affiliation: Department of Molecular Neurobiology, Max Planck Institute of Experimental Medicine, 37075 Göttingen, Germany.
authors.forename: Kerstin
authors.initials: K
authors.lastname: Reim
authors.affiliation: Molecular Biology of Cochlear Neurotransmission Group, InnerEarLab, Department of Otolaryngology, University Medical Center Göttingen, 37075 Göttingen, Germany.
authors.forename: Ellen
authors.initials: E
authors.lastname: Reisinger
authors.affiliation: Department of Molecular Neurobiology, Max Planck Institute of Experimental Medicine, 37075 Göttingen, Germany Collaborative Research Center 889, University of Göttingen, 37099 Göttingen, Germany Center for Nanoscale Microscopy and Molecular Physiology of the Brain, University of Göttingen, 37073 Göttingen, Germany.
authors.forename: Nils
authors.initials: N
authors.lastname: Brose
authors.affiliation: Neurophysiology Group, Department of Molecular Neurobiology, Max Planck Institute of Experimental Medicine, 37075 Göttingen, Germany.
authors.forename: Jeong-Seop
authors.initials: JS
authors.lastname: Rhee
authors.affiliation: Institute for Auditory Neuroscience and InnerEarLab, Department of Otolaryngology, University Medical Center Göttingen, 37099 Göttingen, Germany Collaborative Research Center 889, University of Göttingen, 37099 Göttingen, Germany Center for Nanoscale Microscopy and Molecular Physiology of the Brain, University of Göttingen, 37073 Göttingen, Germany christian.vogl@med.uni-goettingen.de tmoser@gwdg.de carolin.wichmann@med.uni-goettingen.de.
authors.forename: Tobias
authors.initials: T
authors.lastname: Moser
authors.affiliation: Institute for Auditory Neuroscience and InnerEarLab, Department of Otolaryngology, University Medical Center Göttingen, 37099 Göttingen, Germany Collaborative Research Center 889, University of Göttingen, 37099 Göttingen, Germany Molecular Architecture of Synapses Group, Institute for Auditory Neuroscience and InnerEarLab, University Medical Center Göttingen, 37099 Göttingen, Germany christian.vogl@med.uni-goettingen.de tmoser@gwdg.de carolin.wichmann@med.uni-goettingen.de.
authors.forename: Carolin
authors.initials: C
authors.lastname: Wichmann
mesh_terms: D000818:Animals; D002135:Calcium-Binding Proteins / Q000235:genetics; D055032:Electron Microscope Tomography; D016057:Evoked Potentials, Auditory, Brain Stem / Q000502:physiology*; D005089:Exocytosis / Q000502:physiology; D005260:Female; D006199:Hair Cells, Auditory, Inner / Q000166:cytology / Q000378:metabolism*; D006309:Hearing / Q000235:genetics / Q000502:physiology; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics; D008297:Male; D008565:Membrane Proteins / Q000235:genetics*; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D009419:Nerve Tissue Proteins / Q000235:genetics; D009924:Organ Culture Techniques; D018408:Patch-Clamp Techniques; D009435:Synaptic Transmission / Q000502:physiology*; D013572:Synaptic Vesicles / Q000378:metabolism*
chemical_list: C480975:CAPS2 protein, mouse; C510820:Cadps protein, mouse; D002135:Calcium-Binding Proteins; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; C503321:Unc13a protein, mouse; C485549:Unc13b protein, mouse; C485550:Unc13c protein, mouse; C516233:otoferlin protein, mouse
keywords: CAPS; Munc13; Otoferlin; Priming; Ribbon synapse; Tether