CaBP1 and 2 enable sustained CaV1.3 calcium currents and synaptic transmission in inner hair cells
- Experimental Otology Group, InnerEarLab, Department of Otolaryngology, University Medical Center Göttingen, Germany
- Auditory Neuroscience Group, Max Planck Institute for Multidisciplinary Sciences, Germany
- Auditory Systems Physiology Group, Institute for Auditory Neuroscience, InnerEarLab, University Medical Center Göttingen, Germany
- Collaborative Research Center 889, University of Göttingen, Germany
- Functional Auditory Genomics, Institute for Auditory Neuroscience, University Medical Center Göttingen, Germany
- Institute for Auditory Neuroscience and InnerEarLab, University Medical Center Göttingen, Germany
- Restorative Cochlear Genomics Group, Auditory Neuroscience and Optogenetics Laboratory, German Primate Cente, Germany
- Multiscale Bioimaging Cluster of Excellence (MBExC), University of Göttingen, Germany
Figures
Figure 1 with 1 supplement
Strong inactivation of inner hair cell (IHC) CaV1.3 channels upon deletion of Cabp1 and Cabp2.
(A) Schematic of the IHC and one of its several ribbon synapses. CaBPs and CaM modulate voltage-gated calcium channels at the IHC ribbon synapses to shape the presynaptic Ca2+ signal. (B) Peak …
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Figure 1—source data 1
Traces and values obtained from patch-clamp recordings and custom-written macros that were used for analyses in Figure 1B–D, Figure 2, and Figure 1—figure supplement 1.
- https://cdn.elifesciences.org/articles/93646/elife-93646-fig1-data1-v1.zip
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Figure 1—source data 2
Traces and values obtained from patch-clamp recordings and custom-written macros that were used for analyses in Figure 1E, F.
- https://cdn.elifesciences.org/articles/93646/elife-93646-fig1-data2-v1.zip
Figure 1—figure supplement 1
Partial rescue of CaV1.3 channel inactivation properties upon transgenic expression of CaBP2.
(A) The viral vector contains a hCMV/HBA (human cytomegalovirus immediate early enhancer, human beta actin promotor) hybrid promotor to drive strong expression of CaBP2 and a P2A sequence for …
Figure 2
Voltage- and Ca2+-dependent inactivation (VDI and CDI) are enhanced after deletion of Cabp1 and Cabp2.
(A, B) Peak normalized Ca2+ and Ba2+ currents in WT and CaBP1/2-deficient inner hair cells (IHCs) recorded in the extracellular solution containing 2 mM of the respective divalent cation. The two …
Figure 3
Reduced sustained component of exocytosis and calcium charge transfer (QCa) can be partially rescued by Cabp2-transgene expression.
(A) Capacitance increments with the corresponding QCa were probed by different depolarization durations from a holding potential of –85 mV (unpaired t-tests with Welch correction). CaBP1/2-deficient …
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Figure 3—source data 1
Values obtained from patch-clamp exocytosis experiments that were used in Figure 3.
- https://cdn.elifesciences.org/articles/93646/elife-93646-fig3-data1-v1.xlsx
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Figure 3—source data 2
Representative traces and values obtained from patch-clamp exocytosis experiments and custom-written macros that were used for analyses in Figure 3.
- https://cdn.elifesciences.org/articles/93646/elife-93646-fig3-data2-v1.zip
Figure 4
The speed of recovery of Ca2+ currents is affected in CaBP1/2-deficient inner hair cells (IHCs).
(A) Ca2+ currents were probed by 5-ms long depolarization steps to the maximum current potential. An initial test pulse was followed by a conditioning stimulus (a 500-ms depolarization step or a …
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Figure 4—source data 1
Traces and values obtained from patch-clamp recovery experiments and custom-written macros that were used for analyses in Figure 4.
- https://cdn.elifesciences.org/articles/93646/elife-93646-fig4-data1-v1.zip
Figure 5
Comparable synapse density with changed morphology in Cabp1/2-DKO inner hair cells (IHCs).
(A) CaV1.3-immunofluorescence appeared normal in Cabp1/2-DKO-mice. Scale bars: 5 µm. (B) Ribbon synapse immunostainings using RibeyeA as a presynaptic and Homer1 as a postsynaptic marker. Ribbon …
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Figure 5—source data 1
Values obtained from inner hair cell (IHC) ribbon synapse images that were used for analyses in Figure 5G.
- https://cdn.elifesciences.org/articles/93646/elife-93646-fig5-data1-v1.xlsx
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Figure 5—source data 2
Values obtained from inner hair cell (IHC) BK channel images that were used for analyses in Figure 5H.
- https://cdn.elifesciences.org/articles/93646/elife-93646-fig5-data2-v1.xlsx
Figure 6 with 2 supplements
AAV-mediated transgene-expression of Cabp2 ameliorates hearing loss and recovers auditory brainstem response (ABR) amplitude responses in Cabp1/2-DKO mice.
(A) Significant improvement of ABR thresholds in the injected ears of 3- to 4-week-old Cabp1/2-DKO animals for all tone burst frequencies tested as well as 20-Hz-click stimulus (asterisks, see Table …
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Figure 6—source data 1
Values obtained from auditory brainstem response (ABR) recordings that were used for analyses in Figure 6A–C, Figure 6—figure supplement 1A, B, and Figure 6—figure supplement 2C, D.
- https://cdn.elifesciences.org/articles/93646/elife-93646-fig6-data1-v1.xls
Figure 6—figure supplement 1
Auditory brainstem response (ABR) amplitudes are strongly decreased while distortion product otoacoustic emission (DPOAE) are mostly preserved in the Cabp1/2-DKO animals.
(A) ABR thresholds to tone burst and click stimulation show an age-progressive pantonal severe hearing loss in Cabp1/2-DKO mice (asterisks: Šidak’s multicomparison tests for tone burst, Student’s t …
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Figure 6—figure supplement 1—source code 1
Matlab codes used to analyse DPOAE data.
- https://cdn.elifesciences.org/articles/93646/elife-93646-fig6-figsupp1-code1-v1.zip
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Figure 6—figure supplement 1—source data 1
Values obtained from DPOAE recordings that were used for analyses in Figure 6—figure supplement 1C and E.
- https://cdn.elifesciences.org/articles/93646/elife-93646-fig6-figsupp1-data1-v1.xlsx
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Figure 6—figure supplement 1—source data 2
Values obtained from DPOAE recordings that were used for analyses in Figure 6—figure supplement 1D and F.
- https://cdn.elifesciences.org/articles/93646/elife-93646-fig6-figsupp1-data2-v1.xlsx
Figure 6—figure supplement 2
Efficient transgene expression of Cabp2 recovers cumulative auditory brainstem response (ABR) amplitude responses and ABR wave I latencies in Cabp1/2-DKO mice.
(A) Representative images of the organ of Corti of the cochlea immunostained with antibodies against parvalbumin-alpha as a hair cell marker and eGFP. Note a strong eGFP immunofluorescence detected …
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Figure 6—figure supplement 2—source code 1
Matlab source code for GFP fluorescence analysis.
- https://cdn.elifesciences.org/articles/93646/elife-93646-fig6-figsupp2-code1-v1.zip
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Figure 6—figure supplement 2—source data 1
Values obtained from eGFP immunostainings that were used for analyses in Figure 6—figure supplement 2B.
- https://cdn.elifesciences.org/articles/93646/elife-93646-fig6-figsupp2-data1-v1.zip
Figure 7
Impaired sound encoding in Cabp1/2-DKO mice.
Single neuron responses to sound stimulation in vivo in the region of the auditory nerve of Cabp1/2-DKO mice were extremely scarce. (A) WT poststimulus time histograms of spiral ganglion neurons …
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Figure 7—source code 1
Matlab source codes for analysis of SU data.
- https://cdn.elifesciences.org/articles/93646/elife-93646-fig7-code1-v1.zip
Tables
Table 1
Ca2+-current recovery fit parameters.
The data was fitted with the single or the double exponential with the following equations: y(t) = y0 − A1 × exp(−(t/τ1)) and y(t) = y0 − A1 × exp(−(t/τ1)) − A2 × exp(−(t/τ2)), where y(t) represents …
| 500-ms depolarization | 2-min sine wave | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Test group | A1 (%) | τ1 (s) | A2 (%) | τ2 (s) | y0 (%) | A1 (%) | τ1 (s) | A2 (%) | τ2 (s) | y0 (%) |
| DKO | 65 | 0.62 | - | - | 99 | 19 | 1.36 | 21 | 19.41 | 98 |
| WT | 28 | 0.75 | - | - | 100 | 24 | 3.83 | - | - | 97 |
| DKO + PHP.eB-Cabp2 | 22 | 0.47 | 14 | 3.87 | 99 | 13 | 1.88 | 18 | 11.56 | 99 |
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Mus musculus) | Cabp1-KO | Kim et al., 2014 | RRID:MGI:5780462 | A KO of Cabp1 used for cross-breeding to obtain Cabp1/2-DKO animals |
| Strain, strain background (Mus musculus) | Cabp2LacZ/LacZ | Picher, 2017 | RRID:MGI:6155766 | A KO of Cabp2 used for cross-breeding to obtain Cabp1/2-DKO animals |
| Other | PHP.eB viral capsid | Addgene plasmid | #103005 | Gift of V. Gradinaru |
| Recombinant DNA reagent | Encoding CaBP2-L | NCBI Reference Sequence | NP_038906.2 | Long isoform of mouse CaBP2 |
| Antibody | anti-Myosin VIIa (Mouse monoclonal) | Santa Cruz Biotechnology | Cat#: sc-74516, RRID:AB_2148626 | 1:200 |
| Antibody | anti-Kca1.1 (Rabbit polyclonal) | Alomone Labs | Cat#: APC-021, RRID:AB_2313725 | 1:200 |
| Antibody | anti-SK2 (Rabbit polyclonal) | Sigma-Aldrich | Cat#: P0483, RRID:AB_260860 | 1:200 |
| Antibody | anti-CaV1.3 (Rabbit polyclonal) | Alomone Labs | Cat#: ACC-005 RRID:AB_2039775 | 1:100 |
| Antibody | anti-CtBP2 (Mouse monoclonal) | BD Biosciences | Cat#: 612044, RRID:AB_399431 | 1:200 |
| Antibody | anti-Homer1 (Chicken polyclonal) | Synaptic Systems | Cat#: 160006, RRID:AB_2631222 | 1:200 |
| Antibody | anti-Synapsin1/2 (Guinea pig polyclonal) | Synaptic Systems | Cat#: 106004, RRID:AB_1106784 | 1:200 |
| Antibody | anti-RibeyeA (Rabbit polyclonal) | Synaptic Systems | Cat#: 192103, RRID:AB_2086775 | 1:200 |
| Antibody | anti-parvalbumin-α (Guinea pig polyclonal) | Synaptic Systems | Cat#: 195004, RRID:AB_2156476 | 1:200 |
| Antibody | anti-GluA3 (Goat polyclonal) | Santa Cruz Biotechnology | Cat#: sc-7612, RRID:AB_2113895 | 1:200 |
| Antibody | anti-GFP (Chicken polyclonal) | Abcam | Cat#: ab13970, RRID:AB_300798 | 1:200 |
| Antibody | anti-chicken Alexa Fluor 488 (Goat polyclonal) | Thermo Fisher Scientific | Cat#: A-11039, RRID:AB_2534096 | 1:200 |
| Antibody | anti-rabbit Alexa Fluor 488 (Goat polyclonal) | Thermo Fisher Scientific | Cat#: A-11008, RRID:AB_143165 | 1:200 |
| Antibody | anti-guinea pig Alexa Fluor 488 (Goat polyclonal) | Thermo Fisher Scientific | Cat#: A-11073, RRID:AB_2534117 | 1:200 |
| Antibody | anti-rabbit Alexa Fluor 568 (Goat polyclonal) | Thermo Fisher Scientific | Cat#: A-11011, RRID:AB_143157 | 1:200 |
| Antibody | anti-chicken Alexa Fluor 568 (Goat polyclonal) | Abcam | Cat#: ab175711, RRID:AB_2827757 | 1:200 |
| Antibody | anti-mouse Alexa Fluor 647 (Goat polyclonal) | Thermo Fisher Scientific | Cat#: A-21236, RRID:AB_2535805 | 1:200 |
| Antibody | anti-mouse Alexa Fluor 633 (Goat polyclonal) | Thermo Fisher Scientific | Cat#: A-21136, RRID:AB_2535775 | 1:200 |
| Antibody | anti-guinea pig Alexa Fluor 633 (Goat polyclonal) | Thermo Fisher Scientific | Cat#: A-21105, RRID:AB_2535757 | 1:200 |
| Antibody | anti-mouse Alexa Fluor 488 (Donkey polyclonal) | Thermo Fisher Scientific | Cat#: A-21202, RRID:AB_141607 | 1:200 |
| Antibody | anti-goat Alexa Fluor 568 (Donkey polyclonal) | Thermo Fisher Scientific | Cat#: A-11057, RRID:AB_2534104 | 1:200 |
| Antibody | anti-rabbit Alexa Fluor 647 (Donkey polyclonal) | Thermo Fisher Scientific | Cat#: A-31573, RRID:AB_2536183 | 1:200 |
| Software, algorithm | Patchers Power Tools | Igor Pro XOP (http://www3.mpibpc.mpg.de/groups/neher/index.php?page=software) | RRID:SCR_001950SCR_001950 | Analysis of patch-clamp data |
| Software, algorithm | ImageJ | http://imagej.nih.gov/ij/ | RRID:SCR_003070 | Image assembly |
| Software, algorithm | Imaris, Bitplane | http://www.bitplane. com/imaris/imaris | RRID:SCR_007370 | Image assembly and analysis |
| Software, algorithm | Microsoft Excel | https://www.microsoft.com/en-gb/ | RRID:SCR_016137 | Data overviews, algorithm |
| Software, algorithm | Fiji | http://fiji.sc | RRID:SCR_002285 | Image assembly |
| Software, algorithm | GraphPad Prism, GraphPad Software | https://graphpad.com | RRID:SCR_002798 | Statistical analyses and graphs |
| Software, algorithm | IgorPro 6, Wavemetrics | http://www.wavemetrics.com/products/igorpro/igorpro.htm | RRID:SCR_000325 | Data analyses, statistical analyses, and graphs |
| Software, algorithm | MATLAB, Mathworks | http://www.mathworks.com/products/matlab/ | RRID:SCR_001622SCR_001622 | Data analyses, statistical analyses, and graphs |
| Software, algorithm | Patchmaster, HEKA, Harvard Bioscience | https://www.heka.com/index.html | - | Data acquisition |
| Software, algorithm | BioSig, TDT | https://www.tdt.com/components/software/ | RRID:SCR_008428 | Data acquisition |
| Software, algorithm | iMSPECTOR, Abberrior Instruments | https://abberior.rocks/superresolution-confocal-systems/software/ | RRID:SCR_015249 | Data acquisition |
Additional files
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Supplementary file 1
Statistical analysis of ABR thresholds and ABR wave I–V latencies.
The table lists the adjusted p-values of the Tukey’s multicomparisons tests upon one-way (20 Hz clicks) or two-way ANOVA (otherwise) for the ABR thresholds and the click-evoked ABR wave I–V latencies. Very low p-values are represented by asterisks: p < 0.001 (***), p < 0.0001 (****).
- https://cdn.elifesciences.org/articles/93646/elife-93646-supp1-v1.docx
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MDAR checklist
- https://cdn.elifesciences.org/articles/93646/elife-93646-mdarchecklist1-v1.pdf
