title: Rab3-interacting molecules 2α and 2β promote the abundance of voltage-gated CaV1.3 Ca2+ channels at hair cell active zones.
abstract: Ca(2+) influx triggers the fusion of synaptic vesicles at the presynaptic active zone (AZ). Here we demonstrate a role of Ras-related in brain 3 (Rab3)-interacting molecules 2α and β (RIM2α and RIM2β) in clustering voltage-gated CaV1.3 Ca(2+) channels at the AZs of sensory inner hair cells (IHCs). We show that IHCs of hearing mice express mainly RIM2α, but also RIM2β and RIM3γ, which all localize to the AZs, as shown by immunofluorescence microscopy. Immunohistochemistry, patch-clamp, fluctuation analysis, and confocal Ca(2+) imaging demonstrate that AZs of RIM2α-deficient IHCs cluster fewer synaptic CaV1.3 Ca(2+) channels, resulting in reduced synaptic Ca(2+) influx. Using superresolution microscopy, we found that Ca(2+) channels remained clustered in stripes underneath anchored ribbons. Electron tomography of high-pressure frozen synapses revealed a reduced fraction of membrane-tethered vesicles, whereas the total number of membrane-proximal vesicles was unaltered. Membrane capacitance measurements revealed a reduction of exocytosis largely in proportion with the Ca(2+) current, whereas the apparent Ca(2+) dependence of exocytosis was unchanged. Hair cell-specific deletion of all RIM2 isoforms caused a stronger reduction of Ca(2+) influx and exocytosis and significantly impaired the encoding of sound onset in the postsynaptic spiral ganglion neurons. Auditory brainstem responses indicated a mild hearing impairment on hair cell-specific deletion of all RIM2 isoforms or global inactivation of RIM2α. We conclude that RIM2α and RIM2β promote a large complement of synaptic Ca(2+) channels at IHC AZs and are required for normal hearing.
authors.affiliation: Institute for Auditory Neuroscience and InnerEarLab, University Medical Center Göttingen, 37099 Göttingen, Germany; Center for Nanoscale Microscopy and Molecular Physiology of the Brain, University of Göttingen, 37099 Göttingen, Germany;
authors.forename: Sangyong
authors.initials: S
authors.lastname: Jung
authors.affiliation: Institute for Auditory Neuroscience and InnerEarLab, University Medical Center Göttingen, 37099 Göttingen, Germany; Sensory and Motor Neuroscience Program, Göttingen Graduate School for Neurosciences, Biophysics and Molecular Biosciences, University of Göttingen, 37099 Göttingen, Germany;
authors.forename: Tomoko
authors.initials: T
authors.lastname: Oshima-Takago
authors.affiliation: Sensory and Motor Neuroscience Program, Göttingen Graduate School for Neurosciences, Biophysics and Molecular Biosciences, University of Göttingen, 37099 Göttingen, Germany; Molecular Architecture of Synapses Group, Institute for Auditory Neuroscience and InnerEarLab, University Medical Center Göttingen, 37099 Göttingen, Germany; Collaborative Sensory Research Center 889, University of Göttingen, 37099 Göttingen, Germany;
authors.forename: Rituparna
authors.initials: R
authors.lastname: Chakrabarti
authors.affiliation: Institute for Auditory Neuroscience and InnerEarLab, University Medical Center Göttingen, 37099 Göttingen, Germany; Sensory and Motor Neuroscience Program, Göttingen Graduate School for Neurosciences, Biophysics and Molecular Biosciences, University of Göttingen, 37099 Göttingen, Germany; Collaborative Sensory Research Center 889, University of Göttingen, 37099 Göttingen, Germany;
authors.forename: Aaron B
authors.identifier: http://orcid.org/0000-0003-1650-2710
authors.initials: AB
authors.lastname: Wong
authors.affiliation: Sensory and Motor Neuroscience Program, Göttingen Graduate School for Neurosciences, Biophysics and Molecular Biosciences, University of Göttingen, 37099 Göttingen, Germany; Collaborative Sensory Research Center 889, University of Göttingen, 37099 Göttingen, Germany; Auditory Systems Physiology Group, InnerEarLab, Department of Otolaryngology, University of Göttingen Medical Center, 37099 Göttingen, Germany;
authors.forename: Zhizi
authors.initials: Z
authors.lastname: Jing
authors.affiliation: Sensory and Motor Neuroscience Program, Göttingen Graduate School for Neurosciences, Biophysics and Molecular Biosciences, University of Göttingen, 37099 Göttingen, Germany; Collaborative Sensory Research Center 889, University of Göttingen, 37099 Göttingen, Germany; Auditory Systems Physiology Group, InnerEarLab, Department of Otolaryngology, University of Göttingen Medical Center, 37099 Göttingen, Germany;
authors.forename: Gulnara
authors.initials: G
authors.lastname: Yamanbaeva
authors.affiliation: Sensory and Motor Neuroscience Program, Göttingen Graduate School for Neurosciences, Biophysics and Molecular Biosciences, University of Göttingen, 37099 Göttingen, Germany; Collaborative Sensory Research Center 889, University of Göttingen, 37099 Göttingen, Germany; Bernstein Center for Computational Neuroscience, University of Göttingen, 37073 Göttingen, Germany;
authors.forename: Maria Magdalena
authors.initials: MM
authors.lastname: Picher
authors.affiliation: Department of Molecular Neurobiology, Max Planck Institute for Experimental Medicine, 37075 Göttingen, Germany;
authors.forename: Sonja M
authors.initials: SM
authors.lastname: Wojcik
authors.affiliation: Sensory and Motor Neuroscience Program, Göttingen Graduate School for Neurosciences, Biophysics and Molecular Biosciences, University of Göttingen, 37099 Göttingen, Germany; Department of Nanobiophotonics, Max Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany;
authors.forename: Fabian
authors.initials: F
authors.lastname: Göttfert
authors.affiliation: Institute for Auditory Neuroscience and InnerEarLab, University Medical Center Göttingen, 37099 Göttingen, Germany;
authors.forename: Friederike
authors.initials: F
authors.lastname: Predoehl
authors.affiliation: Institute of Neuropathology, University of Bonn, 53111 Bonn, Germany; Department of Epileptology, University of Bonn, 53111 Bonn, Germany.
authors.forename: Katrin
authors.initials: K
authors.lastname: Michel
authors.affiliation: Center for Nanoscale Microscopy and Molecular Physiology of the Brain, University of Göttingen, 37099 Göttingen, Germany; Department of Nanobiophotonics, Max Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany;
authors.forename: Stefan W
authors.initials: SW
authors.lastname: Hell
authors.affiliation: Institute of Neuropathology, University of Bonn, 53111 Bonn, Germany; Department of Epileptology, University of Bonn, 53111 Bonn, Germany.
authors.forename: Susanne
authors.initials: S
authors.lastname: Schoch
authors.affiliation: Collaborative Sensory Research Center 889, University of Göttingen, 37099 Göttingen, Germany; Auditory Systems Physiology Group, InnerEarLab, Department of Otolaryngology, University of Göttingen Medical Center, 37099 Göttingen, Germany; tmoser@gwdg.de NStrenzke@med.uni-goettingen.de cwichma@gwdg.de.
authors.forename: Nicola
authors.initials: N
authors.lastname: Strenzke
authors.affiliation: Molecular Architecture of Synapses Group, Institute for Auditory Neuroscience and InnerEarLab, University Medical Center Göttingen, 37099 Göttingen, Germany; Collaborative Sensory Research Center 889, University of Göttingen, 37099 Göttingen, Germany; tmoser@gwdg.de NStrenzke@med.uni-goettingen.de cwichma@gwdg.de.
authors.forename: Carolin
authors.initials: C
authors.lastname: Wichmann
authors.affiliation: Institute for Auditory Neuroscience and InnerEarLab, University Medical Center Göttingen, 37099 Göttingen, Germany; Center for Nanoscale Microscopy and Molecular Physiology of the Brain, University of Göttingen, 37099 Göttingen, Germany; Collaborative Sensory Research Center 889, University of Göttingen, 37099 Göttingen, Germany; Bernstein Center for Computational Neuroscience, University of Göttingen, 37073 Göttingen, Germany; tmoser@gwdg.de NStrenzke@med.uni-goettingen.de cwichma@gwdg.de.
authors.forename: Tobias
authors.initials: T
authors.lastname: Moser
mesh_terms: D000818:Animals; D020746:Calcium Channels, L-Type / Q000378:metabolism*; D020013:Calcium Signaling; D055032:Electron Microscope Tomography; D016057:Evoked Potentials, Auditory, Brain Stem; D005089:Exocytosis; D006199:Hair Cells, Auditory, Inner / Q000378:metabolism* / Q000648:ultrastructure; D006309:Hearing / Q000502:physiology; D015640:Ion Channel Gating; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D017084:Otoacoustic Emissions, Spontaneous; D018408:Patch-Clamp Techniques; D013136:Spiral Ganglion / Q000378:metabolism; D013569:Synapses / Q000378:metabolism / Q000648:ultrastructure; D013572:Synaptic Vesicles / Q000378:metabolism; D020692:rab3 GTP-Binding Proteins / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*
chemical_list: C485995:Cacna1d protein, mouse; D020746:Calcium Channels, L-Type; C475524:Rim2 protein, mouse; D020692:rab3 GTP-Binding Proteins
keywords: Ca2+ channel; Rab3-interacting molecule; active zone; ribbon synapse; vesicle