CaBP1 and 2 enable sustained CaV1.3 calcium currents and synaptic transmission in inner hair cells

  1. Experimental Otology Group, InnerEarLab, Department of Otolaryngology, University Medical Center Göttingen, 37075 Göttingen, Germany
  2. Auditory Neuroscience Group, Max Planck Institute for Multidisciplinary Sciences, 37075 Göttingen, Germany
  3. Auditory Systems Physiology Group, Institute for Auditory Neuroscience, InnerEarLab, University Medical Center Göttingen, 37075 Göttingen, Germany
  4. Collaborative Research Center 889, University of Göttingen, 37075 Göttingen, Germany
  5. Functional Auditory Genomics, Institute for Auditory Neuroscience, University Medical Center Göttingen, 37075 Göttingen, Germany
  6. Institute for Auditory Neuroscience and InnerEarLab, University Medical Center Göttingen, 37075 Göttingen, Germany
  7. Restorative Cochlear Genomics Group, Auditory Neuroscience and Optogenetics Laboratory, German Primate Center, 37075 Göttingen, Germany
  8. Multiscale Bioimaging Cluster of Excellence (MBExC), University of Göttingen, 37075 Göttingen, Germany

Peer review process

Not revised: This Reviewed Preprint includes the authors’ original preprint (without revision), an eLife assessment, and public reviews.

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Editors

  • Reviewing Editor
    Henrique von Gersdorff
    Oregon Health and Science University, Portland, United States of America
  • Senior Editor
    Andrew King
    University of Oxford, Oxford, United Kingdom

Reviewer #1 (Public Review):

Summary:
This manuscript dissects the contribution of the CaBP 1 and 2 on the calcium current in the cochlear inner hair cells. The authors measured the calcium current inactivation from the double knock-out CaBP1 and 2 and showed that both proteins contribute to voltage-dependent and calcium-dependent inactivation. Synaptic release was reduced in the double KO. As a consequence, the authors observed a depressed activity within the auditory nerve. Taken together, this study identifies a new player that regulates the stimulation-secretion coupling in the auditory sensory cells.

Strengths:
In this study, the authors bring compelling evidence that CaBP 1 and 2 are both involved in the inactivation of the calcium current, from cellular up to system level, and by taking care to probe different experimental conditions such as different holding potentials and by rescuing the phenotype with the re-expression of CaBP2. Indeed, while changing the holding potential worsens the secretion, it completely changes the kinetics of the inactivation recovery. It alerts the reader that probing different experimental conditions that may be closer to physiology is better suited to uncovering any deleterious phenotype. This gave pretty solid results.

Weaknesses:
Although this study clearly points out that CaBP1 is involved in the calcium current inactivation, it is not clear how CaBP1 and CaBP2 act together (but this is probably beyond the scope of the study). Another point is that the authors re-express CaBP2 to largely rescue the phenotype in the double KO but no data are available to know whether the re-expression of both CaBP1 and CaBP2 would achieve a full recovery and what would be the effect of the sole re-expression of CaBP1 in the double KO.

Reviewer #2 (Public Review):

Summary:
In the manuscript by Oestreicher et al, the authors use patch-clamp electrophysiology, immunofluorescent imaging of the cochlea, auditory function tests, and single-unit recordings of auditory afferent neurons to probe the unique properties of calcium signaling in cochlear hair cells that allow rapid and sustained neurotransmitter release. The calcium-binding proteins (CaBPs) are thought to modify the inactivation of the Cav1.3 calcium channels in IHCs that initiate vesicle fusion, reducing the calcium-dependent inactivation (CDI) of the channels to allow sustained calcium influx to support neurotransmitter release. The authors use knockout mice of Cabp1 and Cabp2 in a double knockout (Cabp1/2 DKO) to show that these molecules are required for enabling sustained calcium currents by reducing CDI and enabling proper IHC neurotransmitter release. They further support their evidence by re-introducing Cabp2 using an injection of AAV containing the Cabp2 sequence into the cochlea, which restores some of the auditory function and reduces CDI in patch-clamp recordings.

Strengths:
Overall the data is convincing that Cabp1/2 is required for reducing CDI in cochlear hair cells, allowing their sustained neurotransmitter release and sound encoding. Figures are well-prepared, recordings are careful and stats are appropriate, and the manuscript is well-written. The discussion appropriately considers aspects of the data that are not yet explained and await further experimentation.

Weaknesses:
There are some sections of the manuscript that pool data from different experiments with slightly different conditions (wt data from a previous paper, different calcium concentrations, different holding voltages, tones vs clicks, etc). This makes the work harder to follow and more complicated to explain. However, the major conclusion, that cabp1 and 2 work together to reduce calcium-dependent inactivation of L-type calcium channels in cochlear inner hair cells, still holds.

Another weakness is that the authors used injections of AAV-containing sequences for Cabp2, but do not present data from sham surgeries. In most cases, the improvement of hearing function with AAV injection is believable and should be attributed to the cabp2 function. However, in at least one instance (Figure 4B), the results of the AAV injection experiments may be overinterpreted - the authors show that upon AAV injection, the hair cells have a much longer calcium current recovery following a large, long depolarization to inactivate the calcium channels. Without comparison to sham surgery, it is not known if this result could be a subtle result of the surgery or indeed due to the Cabp2 expression.
It would be great to see the auditory nerve recordings in AAV-injected animals that have a recovery of ABRs. However, this is a challenging experiment that requires considerable time and resources, so is not required.

Reviewer #3 (Public Review):

Summary:
The authors attempted to unravel the role of the Ca2+-binding proteins CaBP1 and CaBP2 for the hitherto enigmatic lack of Ca2+-dependent inactivation of Ca2+ currents in sensory inner hair cells (IHCs). As Ca2+ currents through Cav1.3 channels are crucial for exocytosis, the lack of inactivation of those Ca2+ currents is essential for the indefatigable sound encoding by IHCs. Using a deaf mouse model lacking both CaBP1 and CaBP2, the authors convincingly demonstrate that both CaBP1 and CaBP2 together confer a lack of inactivation, with CaBP2 being far more effective. This is surprising given the mild phenotype of the single knockouts, which has been published by the authors before. Re-admission of CaBP2 through viral gene transfer into the inner ear of double-knockout mice largely restored hearing function, normal Ca2+ current properties, and exocytosis.

Strengths:
1. In vitro electrophysiology: perforated patch-clamp recordings of Ca2+/Ba2+ currents of inner hair cells (IHCs) from 3-4 week-old mice - very difficult recordings - necessary to not interfere with intracellular Ca2+ buffers, including CaBP1 and CaBP2.
2. Capacitance (exocytosis) recordings from IHCs in perforated patch mode.
3. The insight that a negative holding potential might underestimate the impact of lack of CaBP1/2 on the inactivation of ICa in IHCs. As the physiological holding potential is much more positive than a preferred holding potential in patch clamp experiments it has a strong impact on inactivation in the pauses between depolarization mimicking receptor potentials.
This truly advances our thinking about the stimulation of IHCs and accumulating inactivation of the Cav1.3 channels.
4. Insight that the voltage sine method with usual voltage excursions (35 mV) to determine the membrane capacitance (for exocytosis measurements) also favors the inactivated state of Cav1.3 channels
5. Use of double ko mice (for both CaBP1 and CaBP2, DKO) and use of DKO with virally injected CaBP2-eGFP into the inner ear.
6. Use of DKO animals/IHCs/SGNs after virus-mediated CaBP2 gene transfer shows a great amount of rescue of the normal ICa inactivation phenotype.
7. In vivo measurements of SGN AP responses to sound, which is highly demanding.
8. In vivo measurements of hearing thresholds, DPOAE characteristics, and ABR wave I amplitudes/latencies of DKO mice and DKO+injected mice compared to WT mice.

Very thorough analysis and presentation of the data, excellent statistical analysis.

The authors achieved their aims. Their results fully support their conclusions. The methods used by the authors are state-of-the-art.

The impacts on the field are the following:
Regulation of inactivation of Cav1.3 currents is crucial for the persistent functioning of Cav1.3 channels in sensory transduction.
The findings of the authors better explain the phenotype of the human autosomal recessive DFNB93, which is based on the malfunction of CaBP2.
Future work - by the authors or others - should address the molecular mechanisms of the interaction of CaBP1 and 2 in regulating Cav1.3 inactivation.

Weaknesses:
I do not see weaknesses.
What is not explained (but was not the aim of the authors) is how the CaBPs 1 and 2 interact with the Cav1.3 channels and with each other to reduce CDI. Also, why DFNB93, which is based on mutation of the CaBP2 gene, lead to a severe phenotype in humans in contrast to the phenotype of the CaBP2 ko mouse.

  1. Howard Hughes Medical Institute
  2. Wellcome Trust
  3. Max-Planck-Gesellschaft
  4. Knut and Alice Wallenberg Foundation