Disruption of the Presynaptic Cytomatrix Protein Bassoon Degrades Ribbon Anchorage, Multiquantal Release, and Sound Encoding at the Hair Cell Afferent Synapse

Jing, Zhizi; Rutherford, Mark A.; Takago, Hideki; Frank, Tom; Fejtová, Anna; Khimich, Darina; Moser, Tobias; Strenzke, Nicola

doi:10.1523/jneurosci.3491-12.2013

Cited by 121 publications

(163 citation statements)

References 53 publications

(41 reference statements)

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“…In addition, RIM2 may have a role in Ca V 1.3 trafficking to the plasma membrane, but the extent to which this mechanism contributes is less clear, given that palmitoylated Ca V β2a, which is likely expressed in IHCs (35), might take this role. Unlike in Bassoon-deficient IHCs that primarily lack the population of AZs with a large Ca 2+ channel complement (2,9), the cumulative distribution function of Ca V 1.3 immunofluorescence intensities of RIM2α SKO AZs is overall shifted toward smaller values. The shape of the Ca 2+ channel clusters at RIM2α SKO IHC AZs remained stripe-like, indicating that channel localization at the AZ is maintained on the tens of nanometer scale, whereas clusters are disintegrated into two to three round spots on Bassoon-disruption.…”

Section: Discussionmentioning

confidence: 75%

“…Disrupting the presynaptic scaffold protein Bassoon diminishes the numbers of Ca 2+ channels and membrane-tethered vesicles at the AZ (2,8). However, the loss of Bassoon is accompanied by the loss of the entire synaptic ribbon, which makes it challenging to distinguish the direct effects of gene disruption from secondary effects (9).…”

Section: Camentioning

confidence: 99%

“…SGNs were performed as previously described (9,46). In brief, mice aged 4-12 wk were anesthetized by i.p.…”

Section: Recordings Of Auditory Brainstem Response and Distortion Promentioning

confidence: 99%

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Rab3-interacting molecules 2α and 2β promote the abundance of voltage-gated Ca _V 1.3 Ca ²⁺ channels at hair cell active zones

Jung

Oshima-Takago

Chakrabarti

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

169

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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 Ca V 1.3 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 Ca V 1.3 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.ens of Ca V 1.3 Ca 2+ channels are thought to cluster within the active zone (AZ) membrane underneath the presynaptic density of inner hair cells (IHCs) (1-4). They make up the key signaling element, coupling the sound-driven receptor potential to vesicular glutamate release (5-7). The mechanisms governing the number of Ca 2+ channels at the AZ as well as their spatial organization relative to membrane-tethered vesicles are not well understood. Disrupting the presynaptic scaffold protein Bassoon diminishes the numbers of Ca 2+ channels and membrane-tethered vesicles at the AZ (2, 8). However, the loss of Bassoon is accompanied by the loss of the entire synaptic ribbon, which makes it challenging to distinguish the direct effects of gene disruption from secondary effects (9).Among the constituents of the cytomatrix of the AZ, RIM1 and RIM2 proteins are prime candidates for the regulation of Ca 2+ channel clustering and function (10, 11). The family of RIM proteins has seven identified members (RIM1α, RIM1β, RIM2α, RIM2β, RIM2γ, RIM3γ, and RIM4γ) encoded by four genes (RIM1-RIM4). All isoforms contain a C-terminal C 2 domain but differ in the presence of additional domains. RIM1 and RIM2 interact with Ca 2+ channels, most other proteins of the cytomatrix of the AZ, and synaptic vesicle proteins. They interact directly with the auxiliary β (Ca V β) subunits (12, 13) and poreforming Ca V α subun...

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Section: Discussionmentioning

confidence: 75%

Section: Camentioning

confidence: 99%

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Rab3-interacting molecules 2α and 2β promote the abundance of voltage-gated Ca _V 1.3 Ca ²⁺ channels at hair cell active zones

Jung

Oshima-Takago

Chakrabarti

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

169

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“…Single-unit recordings were performed as described previously (43,64,65). In short, mice were anesthetized by i.p.…”

Section: Patch-clampmentioning

confidence: 99%

Ca ²⁺ -binding protein 2 inhibits Ca ²⁺ -channel inactivation in mouse inner hair cells

Picher

Gehrt

Meese

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Ca 2+ -binding protein 2 (CaBP2) inhibits the inactivation of heterologously expressed voltage-gated Ca 2+ channels of type 1.3 (Ca V 1.3) and is defective in human autosomal-recessive deafness 93 (DFNB93). Here, we report a newly identified mutation in CABP2 that causes a moderate hearing impairment likely via nonsense-mediated decay of CABP2-mRNA. To study the mechanism of hearing impairment resulting from CABP2 loss of function, we disrupted Cabp2 in mice (Cabp2 LacZ/LacZ ). CaBP2 was expressed by cochlear hair cells, preferentially in inner hair cells (IHCs), and was lacking from the postsynaptic spiral ganglion neurons (SGNs). Cabp2 LacZ/LacZ mice displayed intact cochlear amplification but impaired auditory brainstem responses. Patch-clamp recordings from Cabp2 LacZ/LacZ IHCs revealed enhanced Ca 2+ -channel inactivation. The voltage dependence of activation and the number of Ca 2+ channels appeared normal in Cabp2 LacZ/LacZ mice, as were ribbon synapse counts. Recordings from single SGNs showed reduced spontaneous and sound-evoked firing rates. We propose that CaBP2 inhibits Ca V 1.3 Ca 2+ -channel inactivation, and thus sustains the availability of Ca V 1.3 Ca 2+ channels for synaptic sound encoding. Therefore, we conclude that human deafness DFNB93 is an auditory synaptopathy.H earing relies on faithful transmission of information at ribbon synapses between inner hair cells (IHCs) and spiral ganglion neurons (SGNs; recently reviewed in refs. 1, 2). Ca 2+ channels at the IHC presynaptic active zone are key signaling elements because they couple the sound-evoked IHC receptor potential to the release of glutamate. IHC Ca 2+ -channel complexes are known to contain Ca V 1.3 α1 subunit (Cav1.3α1) (3-5), betasubunit 2 (Ca V β2) (6), and alpha2-delta subunit 2 (α2δ2) (7) to activate at around −60 mV (8-10), and are partially activated already at the IHC resting potential in vivo [thought to be between −55 and −45 mV (11, 12)], thereby mediating "spontaneous" glutamate release during silence (13).Compared with Ca V 1.3 channels studied in heterologous expression systems, Ca V 1.3 channels in IHCs show little inactivation, which has been attributed to inhibition of calmodulin-mediated Ca 2+ -dependent inactivation (CDI) (14-17) by Ca 2+ -binding proteins (CaBPs) (18,19) and/or the interaction of the distal and proximal regulatory domains of the Ca V 1.3α1 C terminus (20)(21)(22). This "noninactivating" phenotype of IHC Ca V 1.3 enables reliable excitation-secretion coupling during ongoing stimulation (23-25). In fact, postsynaptic spike rate adaptation during ongoing sound stimulation is thought to reflect primarily presynaptic vesicle pool depletion, with minor contributions of Ca V 1.3 inactivation or AMPA-receptor desensitization (23-26). CaBPs are calmodulin-like proteins that use three functional out of four helix-loop-helix domains (EF-hand) for Ca 2+ binding (27). They are thought to function primarily as signaling proteins (28) and differentially modulate calmodulin effectors (29,30). In addition, CaBPs m...

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“…The following antibodies were used: mouse anti-βIII tubulin (R&D systems, MAB1195, clone #TuJ1, 1/400), rabbit anti-ryanodine receptor (Braubach et al, 2014) (Millipore, AB9078, 1/200), goat anti-VaChT (Atasoy et al, 2014) (Millipore, ABN100, 1/100), goat anti-ChAT (Sümbül et al, 2014) (Millipore, AB144, 1/100), chicken anti-neurofilament H (NFH) (Wainger et al, 2015) (Millipore, AB5539, 1/400), mouse anti-dihydropyridine receptor (DHPR) (Bradley et al, 2014) (Abcam, Ab2864, 1/400), mouse anti-synaptotagmin (Wong et al, 2014) (Abcam, ab13259 clone ASV30, 1/100), mouse anti-α-actinin (Falcone et al, 2014) (Sigma-Aldrich, A5044 clone BM-75.2, 1/500), mouse anti-sodium channel pan (Bailey et al, 2003) (Sigma-Aldrich, clone K58/35, S8809, 1/100), mouse anti-ankyrin (Bailey et al, 2003) (Thermo Scientific, 33-8800, clone 4G3F8, 1/100), mouse antibassoon (Jing et al, 2013) (Abcam, ab82958, 1/100), rabbit anti-glial fibrillary acidic protein (GFAP) (Achtstätter et al, 1986) (Dako, Z0334, 1/100), mouse anti-oligodendrocytic marker O4 (Paintlia et al, 2004) (Sigma-Aldrich, O7139, clone O4, 1/400), rabbit anti-MuSK (serum T194, gift from Markus Ruegg, Biozentrum, University of Basel, Switzerland, 1/500), mouse anti-rapsyn [Abcam, ab11423 (1234), 1/200], mouse antiSyne1 (clone 8c3, gift from Glenn Morris, Keele University, UK, 1/200).…”

Section: Primary Antibodiesmentioning

confidence: 99%

A system for studying mechanisms of neuromuscular junction development and maintenance

Vilmont¹,

Cadot²,

Ouanounou³

et al. 2016

Journal of Cell Science

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The neuromuscular junction (NMJ), a cellular synapse between a motor neuron and a skeletal muscle fiber, enables the translation of chemical cues into physical activity. The development of this special structure has been subject to numerous investigations, but its complexity renders in vivo studies particularly difficult to perform. In vitro modeling of the neuromuscular junction represents a powerful tool to delineate fully the fine tuning of events that lead to subcellular specialization at the pre-synaptic and post-synaptic sites. Here, we describe a novel heterologous co-culture in vitro method using rat spinal cord explants with dorsal root ganglia and murine primary myoblasts to study neuromuscular junctions. This system allows the formation and long-term survival of highly differentiated myofibers, motor neurons, supporting glial cells and functional neuromuscular junctions with post-synaptic specialization. Therefore, fundamental aspects of NMJ formation and maintenance can be studied using the described system, which can be adapted to model multiple NMJ-associated disorders.

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Disruption of the Presynaptic Cytomatrix Protein Bassoon Degrades Ribbon Anchorage, Multiquantal Release, and Sound Encoding at the Hair Cell Afferent Synapse

Cited by 121 publications

References 53 publications

Rab3-interacting molecules 2α and 2β promote the abundance of voltage-gated Ca _V 1.3 Ca ²⁺ channels at hair cell active zones

Rab3-interacting molecules 2α and 2β promote the abundance of voltage-gated Ca _V 1.3 Ca ²⁺ channels at hair cell active zones

Ca ²⁺ -binding protein 2 inhibits Ca ²⁺ -channel inactivation in mouse inner hair cells

A system for studying mechanisms of neuromuscular junction development and maintenance

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Disruption of the Presynaptic Cytomatrix Protein Bassoon Degrades Ribbon Anchorage, Multiquantal Release, and Sound Encoding at the Hair Cell Afferent Synapse

Cited by 121 publications

References 53 publications

Rab3-interacting molecules 2α and 2β promote the abundance of voltage-gated Ca V 1.3 Ca 2+ channels at hair cell active zones

Rab3-interacting molecules 2α and 2β promote the abundance of voltage-gated Ca V 1.3 Ca 2+ channels at hair cell active zones

Ca 2+ -binding protein 2 inhibits Ca 2+ -channel inactivation in mouse inner hair cells

A system for studying mechanisms of neuromuscular junction development and maintenance

Contact Info

Product

Resources

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Rab3-interacting molecules 2α and 2β promote the abundance of voltage-gated Ca _V 1.3 Ca ²⁺ channels at hair cell active zones

Rab3-interacting molecules 2α and 2β promote the abundance of voltage-gated Ca _V 1.3 Ca ²⁺ channels at hair cell active zones

Ca ²⁺ -binding protein 2 inhibits Ca ²⁺ -channel inactivation in mouse inner hair cells