Rab3-interacting molecules 2α and 2β promote the abundance of voltage-gated CaV1.3 Ca²⁺ channels at hair cell active zones

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 112; no. 24; pp. E3141 - E3149
• Main Authors: Jung, Sangyong, Oshima-Takago, Tomoko, Chakrabarti, Rituparna, Wong, Aaron B., (黃沛燊), Jing, Zhizi, Yamanbaeva, Gulnara, Picher, Maria Magdalena, Wojcik, Sonja M., Göttfert, Fabian, Predoehl, Friederike, Michel, Katrin, Hell, Stefan W., Schoch, Susanne, Strenzke, Nicola, Wichmann, Carolin, Moser, Tobias
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 16.06.2015; National Acad Sciences
• Series: PNAS Plus
• Subjects: Animals; Biological Sciences; brain; calcium; calcium channels; Calcium Channels, L-Type - metabolism; Calcium Signaling; Electron Microscope Tomography; Evoked Potentials, Auditory, Brain Stem; Exocytosis; ganglia; Hair Cells, Auditory, Inner - metabolism; Hair Cells, Auditory, Inner - ultrastructure; hearing; Hearing - physiology; Ion Channel Gating; knockout mutants; Mice; Mice, Inbred C57BL; Mice, Knockout; neurons; Otoacoustic Emissions, Spontaneous; Patch-Clamp Techniques; PNAS Plus; rab3 GTP-Binding Proteins - deficiency; rab3 GTP-Binding Proteins - genetics; rab3 GTP-Binding Proteins - metabolism; Spiral Ganglion - metabolism; Synapses - metabolism; Synapses - ultrastructure; Synaptic Vesicles - metabolism; Rab3-interacting molecule; ribbon synapse; vesicle; Ca2+ channel; active zone
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.1417207112

Ca²⁺ 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 Ca²⁺ 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 Ca²⁺ imaging demonstrate that AZs of RIM2α-deficient IHCs cluster fewer synaptic CaV1.3 Ca²⁺ channels, resulting in reduced synaptic Ca²⁺ influx. Using superresolution microscopy, we found that Ca²⁺ 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 Ca²⁺ current, whereas the apparent Ca²⁺ dependence of exocytosis was unchanged. Hair cell-specific deletion of all RIM2 isoforms caused a stronger reduction of Ca²⁺ 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 Ca²⁺ channels at IHC AZs and are required for normal hearing.