Introduction

Calcium influx through voltage-gated calcium channels (Ca_Vs) is a pivotal initiator of neurotrans-mission. These channels typically undergo considerable Ca²⁺- and voltage-dependent inactivation (CDI and VDI), which limits the amount of incoming Ca²⁺ ions and thus possible excitotoxicity. In sensory receptor cells such as sensory inner hair cells and photoreceptors, however, the extent of inactivation is significantly reduced to allow encoding of graded and sustained membrane potential fluctuations in response to ongoing sound or visual (t.i. “darkness”) stimuli [reviewed in ¹]. Further-more, slowed inactivation of L-type Ca_Vs was also reported at ribbon synapses of retinal bipolar cells ², which transmit visual information via continuous exocytosis of synaptic vesicles onto retinal ganglion cells. The attenuation of channel inactivation may be largely attributed to the action of calcium-binding proteins, CaBPs ³, which primarily abate the calmodulin (CaM)-mediated CDI of Ca_V channels ^3–8. At the inner hair cell (IHC) ribbon synapses, the Ca²⁺ current is carried through the L-type Ca_V1.3 channels ^9,10. Their biophysical properties may be regulated by several CaBP family members ^11,12, but most prominently by CaBP2 ¹³. Mutations in the CaBP2 gene cause moderate to severe autosomal recessive hearing impairment DFNB93 [see e.g. ^13–17]. Interestingly, the loss of CaBP2 seems to significantly affect VDI, but not CDI of the IHC Ca²⁺ currents ¹³. Furthermore, isolated disruption of CaBP1 or CaBP4 in mice leads to at most modest enhancement of IHC Ca_V1.3 inactivation ^11,12. Moreover, the automodulatory C-terminal domain of the long Ca_V1.3 isoform, abundant at the IHC ribbon synapses ^18,19, does not seem critical for CDI suppression either, as its disruption does not lead to hearing impairment ¹⁹. The question of which mechanisms are primarily responsible for suppressing IHC Ca_V1.3 CDI thus remains unresolved. Since CaBP2, followed by the structurally similar CaBP1, are the most abundant members of the CaBP family in the cochlea ²⁰, we set out to investigate the phenotype of Cabp1/2 double-knockout (DKO) animals at the systems and cellular levels. Severe hearing impairment that we observed in these animals reveals that CaBP1 and 2 act together to attenuate the inactivation of the IHC Ca_V1.3 channels. Furthermore, we demonstrate that the combined absence of CaBP1 and 2 almost abolishes sustained, and, upon increased hair cell synaptic activity, the fast component of exocytosis. Such phenotype was observed neither in Cabp1 nor in Cabp2 single KO animals ^12,13. To obtain a more comprehensive understanding of the roles of the two calcium-binding proteins, we attempted to rescue the auditory function of the CaBP1/2-depleted auditory system by viral delivery of a Cabp2 coding sequence in the inner ear. These experiments revealed a substantial recovery of hearing, which may be related to the successful restoration of Ca²⁺ currents and IHC exocytosis upon transgenic expression of CaBP2. Our findings indicate that the functions of CaBP1 and 2 partially overlap at the IHC ribbon synapse. Together, they provide sufficient attenuation of channel inactivation to sustain Ca²⁺ currents and exocytosis required for temporally precise, indefatigable neurotrans-mission at this synapse.

Results

Pronounced VDI and CDI of Ca_V1.3 channels in CaBP1/2-deficient IHCs

CaBP1 and 2 show high expression levels in the organ of Corti ²⁰, where they may modulate voltage-gated Ca_V1.3 channels at the IHC ribbon synapses (Fig. 1A). Deletion of CaBP2 results in an early-onset, progressive hearing impairment in the KO mouse model and moderate to severe hearing impairment, DFNB93, in affected human patients ^13–15. Loss of CaBP1 in mice, on the other hand, leaves hearing unperturbed in the first few months after birth ¹². Since these proteins show a high degree of homology ³, it is plausible that they possess similar features and are partially redundant, at least initially. Therefore, we were interested in understanding their cumulative impact on the function of hair cells and hearing. For this, we analyzed the phenotype of the Cabp1/2-DKO animals and tested the extent of functional rescue following the transgenic expression of CaBP2 (Fig. S1A) in the ears of the DKO mice.

Figure 1.

To investigate possible synergistic functions of the two major IHC CaBPs, we first measured Ca²⁺ currents in the IHCs of 3-4-week-old Cabp1/2-DKO and WT animals in the presence of 1.3 mM extracellular [Ca²⁺]. Employing 500 ms-long depolarization steps to maximal Ca²⁺-current potential (-25 to -15 mV) revealed pronounced inactivation of Ca_V1.3 channels in the DKO IHCs (Fig. 1B). This was significantly stronger when compared to the inactivation observed in the IHCs of Cabp1 or Cabp2 single KOs in similar conditions (Fig. 1C) ^12,13,21, and even when comparing to data from 5-week-old Cabp2 KO mice obtained at physiological temperature (Fig. S1D), further substantiating the evidence of severity and early onset of the DKO phenotype.

The “non-inactivating” nature of the IHC Ca_V1.3 channels was partially restored in the Cabp1/2-DKOs by transgenic expression of WT CaBP2 (Student’s t-test: p < 0.0001; Fig. 1B-C; see also Fig. S1). The lack of complete recovery suggests either an inadequate expression of transgenic material or irreversible adaptive changes in response to the lack of major CaBPs that may occur prior to gene delivery. Similarly, Ca²⁺ currents were greatly reduced upon application of short train stimuli and rescued efficiently by the introduction of the transgenic CaBP2 (Fig. 1D). However, it has to be noted that the cells in which train stimuli were applied showed a considerably lower calcium current inactivation upon a 500-ms step depolarization than the average (0.36 (n=5) vs 0.47 (n=21)). Short stimuli at different potentials revealed maximal Ca²⁺-current amplitudes in the IHCs of DKO mice that were comparable to controls (Fig. 1E). We observed a slight shift in the voltage-dependence of Ca_V1.3 activation towards more hyperpolarized values (Fig. 1F; potential of half-activation – V_0.5 of -33.6 ± 0.5 vs -30.9 ± 0.5 mV in Cabp1/2 DKO and WT IHCs, respectively; p < 0.0001, Student’s t-test), and an increased slope of the Boltzmann fit function (7.6 ± 0.1 vs 7.1 ± 0.1, p < 0.0001, Student’s t-test). This finding is somewhat surprising as the opposite would be expected based on data from heterologous expression systems ^5,13,22. At present we can not exclude potential changes in the relative amounts of different Ca_V1.3 splice variants, auxiliary subunits or other interaction partners that may in the absence of CaBPs lead to these changes in the IHCs.

To further investigate the nature of the enhanced Ca_V1.3 inactivation, we recorded Ca_V1.3 currents in the presence of 2 mM Ca²⁺ or Ba²⁺ in the extracellular solution (Fig. 2), and analyzed the two types of inactivation as described previously ²³. First, VDI was calculated from the Ba²⁺ currents, followed by the calculation of the CDI from the comparison between the Ba²⁺ and Ca²⁺ currents (for mathematical formulas, see Methods). The lack of CaBP1 and 2 significantly enhanced both, VDI and CDI of IHC Ca_V1.3 channels, which were approx. two- and six-times larger than those in WT IHCs (Fig. 2B; VDI: 0.30 ± 0.02 vs 0.13 ± 0.02 and CDI: 0.65 ± 0.01 vs 0.11 ± 0.02 in Cabp1/2 DKO and WT IHCs, respectively; p < 0.0001, Student’s t-test).

Figure 2.

Impaired synaptic transmission in the IHCs lacking CaBP1 and 2

Since Ca²⁺ influx through Ca_V1.3 channels triggers IHC neurotransmission we next investigated how the prominent inactivation affects exocytosis in the CaBP1/2-deficient IHCs. Exocytosis was probed by recordings of membrane capacitance (Fig. 3). In response to short stimuli [primarily recruiting the readily-releasable pool (RRP) of vesicles], increases in capacitance were comparable, while long stimuli (reporting sustained exocytosis) evoked significantly smaller responses in the CaBP1/2-deficient IHCs (Fig. 3A-B). These results are in line with the observation of comparable peak Ca²⁺-current amplitudes but reduced calcium charge transfer (Q_Ca) for stimulus durations above 50 ms. However, calculating exocytosis efficiency revealed that the reduction in sustained exocytosis cannot completely be explained by the diminished Ca²⁺ influx (Fig. 3C). This suggests an additional impairment in exo- or endocytic processes, the underlying mechanism of which is currently unknown. Transgenic expression of CaBP2 largely restored the efficiency of exocytosis, indicating that the impairment as observed in the IHCs of DKOs is related to the absence of the CaBPs. Such exocytosis defect was not observed in Cabp2-KOs, suggesting that when IHCs are held at hyperpolarized potential at RT, CaBP1 provides sufficient compensation to obscure the synaptic phenotype ^13,21.

Figure 3.

Next, we have reasoned that holding a cell at a relatively hyperpolarized potential of -85 mV as typically utilized in patch-clamp recordings may not reveal the synaptic phenotype in its severity. We thus performed additional sets of experiments (Fig. 3D-E), in which the cells were held at either -65 or -55 mV, closer to the suggested resting potential of IHCs ^24–26. Raising the holding voltage to -55 mV resulted in a significant reduction in the maximal Ca²⁺ currents, which was more pronounced in the CaBP1/2-deficient IHCs as compared to controls (60 ± 4 % vs 25 ± 4 % reduction in DKO and WT IHCs, respectively; p < 0.005, Student’s t-test). Consequently, further discrepancy between the Q_Ca in IHCs of WT and DKO animals was observed (p < 0.001), which in DKO animals resulted in a complete loss of sustained exocytosis (Fig. 3D; p < 0.005). The latter was well restored upon AAV-mediated transgenic expression of CaBP2 (p < 0.05), which also reduced the amount of calcium channel inactivation (38 ± 8 % reduction in the maximal Ca²⁺ current as well as a partial recovery of Q_Ca as compared to Cabp1/2-DKO IHCs; p < 0.05). Rapid voltage oscillations during capacitance recordings may additionally activate calcium channels prior to the depolarizing step. We thus reduced the sine wave amplitude, which resulted in an intermediate phenotype, indicating pronounced activity-dependent deterioration of synaptic function in the absence of CaBPs (Fig. 3E). We propose that in vivo in the absence of CaBP1 and 2 a large fraction of IHC Ca_V1.3 channels are in a state of prolonged inactivation. This does not support sufficient RRP exocytosis (Fig. 3D) to sustain sensory coding during ongoing stimulation of IHCs.

Lastly, we asked whether the deletion of CaBP1 and 2 affects the speed of recovery from Ca_V1.3 inactivation (Fig. 4). We applied an initial 5-ms long pulse followed by a conditioning stimulus and a series of 5-ms long pulses to test the kinetics of recovery of the Ca²⁺-current amplitude. Upon 500-ms depolarization step (Fig. 4B), the speed of recovery from inactivation was fast both in DKO as well as WT IHCs where a much smaller fraction of channels underwent inactivation (time constants of 0.62 and 0.75 s in DKO and WT IHCs, respectively; Table 1, left). As expected, transgenic expression of CaBP2 reduced the initial inactivation (Ca²⁺-current amplitude 10 ms after the conditioning pulse, expressed as the fraction of initial current – I_{10ms_post}/I_pre, WT: 0.73 ± 0.01; DKO: 0.36 ± 0.03; DKO+PHP.eB-Cabp2: 0.64 ± 0.02; p < 0.001 for DKO vs DKO+PHP.eB-Cabp2). However, the recovery from the step-evoked inactivation was slowest in the Cabp2-injected DKO IHCs. We hypothesize that this may be related to the speed at which the intracellular [Ca²⁺] after depolarization is restored to the basal level allowing Ca_V1.3 recovery from CDI (see discussion). A 2-min long sine wave (± 15 mV) from -55 mV to mimic a milder but prolonged hair cell stimulation (Fig. 4C) elicited smaller inactivation of Ca_V1.3 channels as compared to a 500-ms depolarization step (I_{10ms_post}/I_pre of 0.73 ± 0.01 in WT; 0.58 ± 0.03 in DKO; 0.68 ± 0.03 in DKO+PHP.eB-Cabp2; p < 0.05 for DKO vs DKO+PHP.eB-Cabp2). In these conditions, however, the recovery from inactivation was slowest in the DKO IHCs (Table 1, right). This suggests that in the absence of CaBP1 and 2 even mild but prolonged stimulation of IHCs in vivo may drive a significant fraction of Ca_V1.3 channels into “deep-state” of inactivation with very slow recovery, as was described for Na_V, K_V and Ca_V channels ^27–30, not allowing efficient sound encoding.

Figure 4.

Table 1.

Altered synaptic morphology in Cabp1/2-DKO mice

Immunolabeling of the organs of Corti indicated normal presynaptic Ca_V1.3 clustering with no apparent change in size or shape of the clusters (Fig. 5A), and comparable abundance of ribbon synapses in the apical IHCs of WT and DKO animals (12.9 ± 0.2 vs 12.8 ± 0.8; respectively; Fig. 5B) at 3 weeks of age. However, the intensities of pre- (RibeyeA/CtBP2) and postsynaptic (Homer1) immunofluorescent puncta were increased in the CaBP1/2-deficient IHCs as compared to age-matched controls, processed in parallel (Fig. 5G). A similar alteration in the synaptic size was observed in some other synaptic mutants as well as upon noise trauma ^31,32, but not in Cabp2 single KOs ¹³, which show significantly less impaired presynaptic Ca²⁺ signalling. This data is in line with compensatory postsynaptic changes in conditions of no or very little postsynaptic activity, possibly governed through adaptive changes in the postsynaptic elements.

Fig. 5.

As earlier studies of Ca_V1.3 channelopathies demonstrated a lag or stop in IHC development ^31,33,34 we next examined the expression of small-(SK) and large-conductance Ca²⁺-activated potassium channels (BK) in the IHCs at different ages (Fig. 5C-F). As expected for normal maturation, the SK2 puncta were mostly absent from DKO IHCs by 3 weeks of age and the BK puncta underwent a typical transition from a spotty appearance throughout the cell membrane to the patches at the IHC necks (Fig. 5F). However, by 3 weeks and more obviously later on, the BK channel clusters appeared smaller in the DKO IHCs as compared to controls, which could be partially reversed by virally-mediated expression of CaBP2 (Fig. 5E). Using Imaris, we analyzed the respective “volumes” of BK-cluster immunofluorescence in the IHC neck region from ca. 4-week-old mice. This analysis revealed significantly smaller (Fig. 5H; Student’s t-test, p < 0.0001, N=6 organs per genotype) and fewer clusters in DKO IHCs. This was also obvious when comparing the cumulative “volume” of BK cluster immunofluorescence per IHC, which was significantly higher in WT IHCs, indicating higher abundance of BK channels (14.2 ± 2.1 vs 6.6 ± 1.1 µm³ per IHC, Student’s t-test, p < 0.01). We hypothesize that CaBPs may directly (e.g. as transcription factors) regulate BK expression, a notion that needs to be tested in the future. Alternatively, the effects may be more indirect, as they are common in mutants with impaired intracellular Ca²⁺ signaling ^31,33,34.

Severe hearing impairment in mice deficient in CaBP1 and CaBP2

Hearing was assessed by measuring auditory brainstem responses (ABRs; Fig. 6, Fig. S2). Already in young (3-4-week-old) Cabp1/2-DKO animals, ABR thresholds were highly elevated, with tone bursts up to a maximum intensity of 90 dB barely eliciting any ABR response. Click responses were better preserved (thresholds of 59 ± 2 dB in DKO (N=9) vs 30 ± 2 dB in WT (N=8) mice) but greatly reduced in amplitude (Fig. S2A-B). At 7-13 weeks of age, further worsening of the click thresholds was observed (68 ± 5 dB in DKO vs 30 ± 1 dB in WT mice; N=7 for both). Upon 80-dB click stimulation no sizable ABR wave I was detected in average traces of DKO animals at either age tested (Fig. S2B), suggesting reduced and/or desynchronized spiking of spiral ganglion neurons (SGNs). Like in other mouse mutants with defects in the IHC synapse ^35,36, the later ABR peaks were better preserved, indicating partially preserved activation of neurons of the auditory brainstem. To assess cochlear amplification by the outer hair cells (OHCs), we recorded distortion product otoacoustic emissions (DPOAEs). While OAEs were so far only detectable in some of the younger DFNB93 patients ^13,37, mouse work corroborates the notion of at least initially intact cochlear amplification in the absence of CaBP2 ^12,13,21. Our results suggest that cochlear amplification is largely preserved also in DKO animals (Fig. S2C-F). In about half of the DKO animals in both age groups, the DPOAEs were normal. In the other half, they were reduced in amplitude but still detectable (Fig. S2E-F), which may suggest partially hampered cochlear amplification in a subset of DKO animals.

Figure 6.

Transgenic expression of CaBP2 led to significant improvement of the IHC Ca_V1.3 function and synaptic exocytosis (Figs. 1-4), we thus next asked, how this may influence the hearing of the animals. As previously observed ²¹, unilateral injection of the PHP.eB-Cabp2-eGFP resulted in strong eGFP immunofluorescence signal in the vast majority of hair cells and elsewhere (Fig. S3A-B, E). Transgenic expression of CaBP2 in DKO animals led to significantly improved hearing with lower ABR thresholds, increased click-evoked ABR amplitudes, and shortened wave I latency (Fig. 6, Fig. S3C-D).

Finally, we studied sound encoding by inspecting sound-induced spiking responses of SGNs (Fig. 7). In WT mice, we recorded responses in 39 neurons, which were classified as SGNs based on their location, response patterns, spike waveforms and recording stability. Their spiking characteristics resembled typical WT data ^13,38–41. In response to 50- or 500-ms tone burst at the characteristic frequency 30 dB above threshold (i.e. where the sigmoidal rate-intensity functions have saturated spike rates) and at a stimulus repetition rate of 5 Hz, they showed the SGN-typical very high onset firing rates, gradually adapting to reach steady-state rates between 100 and 400 Hz (Fig. 7A-B). In contrast, only one out of the 20 neurons we recorded and analyzed from DKO animals showed a normal response in our automated tuning curve algorithm with a threshold of 35 dB at 7.8 kHz, while all others displayed thresholds above 80 dB and extremely low spike rates (Fig. 7A-B). Since in the majority of neurons frequency tuning was very broad, we used subjectively chosen best frequency tone burst or white noise burst stimulation of at least 80 dB for subsequent characterization. 16 out of 19 DKO units responded with a relatively high onset rate but strong adaptation to a minimal sustained spiking (Fig. 7A-B), which prevented unit classification based on the response patterns. Judging from other criteria described above, we assume that approximately 13 of these represent SGNs and 3 represent bushy cells or multipolar cells of the anteroventral cochlear nucleus. Three additional units (two resembling a pauser/buildup- and one displaying a clear chopper neuron-type response) were excluded from further analysis. Overall, in DKO neurons, peak and adapted rates were greatly reduced as compared to WT controls (Fig. 7C), which came along with an increased latency (12.6 ± 2.8 ms vs WT 4.8 ± 0.2 ms; p < 0.001, Wilcoxon rank-sum test) and increased jitter (18.2 ± 5.0 vs WT 1.5 ± 0.4; p < 0.00001, Wilcoxon rank-sum test) of the first spike that followed the spike onset.

Figure 7.

DKO responses greatly improved when we lowered the stimulus repetition rate to 0.5 Hz, and plateaued at 0.2 Hz (Fig. 7C-D). We hypothesize that in DKO SGNs, recovery of Ca_V1.3 channels after a silent interval in the range of 1-2 seconds is the limiting factor for determining the maximal SGN spiking rate at the sound onset. This may be further contributed by potentially slowed re-supply of synaptic vesicles, as suggested by reduced efficiency of sustained exocytosis in the CaBP1/2-deficient IHCs (see Fig. 3). In contrast, in WT SGNs, the maximal spike rates are mostly recovered after a silent interval of 150 ms, which may be mostly limited by vesicle replenishment at the IHC ribbon synapse ^36,42. During a 5-Hz sound stimulation, the strength of adaptation was greatly enhanced (peak to adapted ratio 12.5 ± 3.0 vs WT 3.4 ± 0.1; p < 0.00001, Wilcoxon rank-sum test) whereas the time constant of adaptation was unchanged (7.9 ± 2.0 ms in DKO compared to 7.0 ± 0.4 ms in WT (p = 0.5, Wilcoxon rank-sum test). However, for the longer 500-ms tone bursts used in the 0.5 Hz stimulation dataset, it becomes obvious that despite the strong adaptation, DKO neurons are still able to sustain spiking at a low rate throughout the stimulation (Fig. 7B). DKOs also showed reduced spontaneous spiking activity (Fig. 7E), which supports the hypothesis that already in the absence of sound stimulation over tens of seconds a considerable fraction of calcium channels may be in steady-state inactivation.

The reduction in spike rates and their strong dependence on the strength of stimulation in the preceding seconds introduces experimental bias to our data. In the units of the DKO animals, it was not feasible to faithfully assess sound thresholds or dynamic ranges. Furthermore, the overall experimental yield was generally much lower in DKO animals. On average we hit 0.3 sound-responsive neurons per hour of recording, compared to 2.5 in WT. Very likely many DKO neurons failed to generate spontaneous and evoked action potentials, thus we were not able to identify them. Based on the DPOAE data we assume that cochlear mechanics and active amplification and thus the IHC receptor potential thresholds are mostly normal in the DKOs. We cannot exclude that IHC receptor potentials are decreased in amplitude, which however should rather reduce SGN adaptation. We thus interpret the observed increase in adaptation and delay of recovery of SGN spiking to be the effects of changed synaptic Ca_V1.3 channel kinetics as described above.

In summary, sound responses of DKO SGNs and cochlear nucleus neurons showed a drastic reduction in spontaneous and evoked action potential rates, which was associated with increased sound thresholds and reduced temporal precision of action potential generation. Together, these factors explain well the poor ABR thresholds (Fig. 6, Fig. S2A).

Discussion

In this study, we examined the effects of genetic disruption of the two major CaBPs expressed in the cochlea. In the IHCs of CaBP1/2-deficient animals, we found largely inactivated Ca²⁺ currents with prolonged recovery from inactivation upon mild oscillatory stimulation, which did not support sufficient neurotransmission for proper sound encoding. The extent of channel inactivation and impairment of IHC synaptic exocytosis aggravated with the extent of IHC stimulation. Similarly, SGN action potential rates in vivo declined at increased sound stimulation rates and displayed very strong adaptation upon stimulation, going along with impaired sustained IHC exocytosis. The impairment was significantly stronger as compared to the phenotype in the absence of CaBP2 alone. This suggests that under in vivo conditions the roles of CaBP1 and 2 are partially overlapping.

Function and expression profile of CaBPs in the cochlea

The murine cochlea shows strong expression of CaBP1 and 2, while the levels of CaBP4 and 5 seem negligible ^20,43,44. Among the three putative splice isoforms of CaBP2 differing in the N-terminal aminoacid sequence, the alternative long isoform, termed CaBP2-alt, may be the most abundant splice variant in the IHCs, as suggested by in situ hybridization ²⁰, while SGNs seem to express none. So far our gene-rescue experiments were conducted with the conventional L-isoform only, which however shows strong inhibition of Ca_V1.3 inactivation in the HEK cells as well as IHCs ^13,21. Alternative splicing also generates three CaBP1 variants ²⁰. The alternative long variant caldendrin is primarily expressed in the SGNs or the surrounding glia cells, while weak expression of CaBP1-S/-L (as judged by the mRNA production) was detected in hair cells ²⁰. Our DKO model lacks all splice isoforms of both CaBPs. Since Cabp1 KO animals show normal hearing several weeks after birth, and CaBP2 is absent from SGNs, we interpret the hearing impairment of the Cabp1/2 DKO animals to be primarily due to defects of hair cell function and judge contribution by SGN dysfunction as rather unlikely, at least in the early postnatal weeks.

The ABR thresholds of Cabp1 KO animals are normal at 4 weeks of age, but deteriorate with age, starting with high-frequency hearing loss at 9 weeks. Conversely, the suprathreshold ABRs to tone burst stimulation show increased amplitudes and reduced latencies during the first two months after birth. This suggested enhanced SGN activity at first, followed by slowly progressing functional decline, possibly due to overexcitability-related toxicity ¹². Due to early strong impairment of hair cell synaptic function, the latter effects are not expected to occur in the DKO animals. However, ABR thresholds for clicks were further increased and an additional reduction in the summating potential was observed in the DKO animals at two as compared to one month of age despite relatively well-preserved DPOAEs (Fig. S2A-D). It thus remains to be clarified whether the absence of CaBP1 and 2 may affect (IHC) mechanotransduction at more mature stage. Unfortunately, this is difficult to assess due to the increased fragility of hair bundles at mature age. Intriguingly, CaBPs share similarities with calcium and integrin-binding protein 2 (CIB2), associated with hair bundle defects ^45–47. When we overexpressed CaBP2 in the DKO animals, interestingly, suprathreshold responses of ABR wave I were very large (Fig. 6) and the summated wave I-IV responses were not distinguishable from WT responses (Fig. S3C). However, tone burst and click thresholds did not completely recover to WT levels. The difference in the extent of rescue of threshold and suprathreshold responses may be due to possibly distinct requirements for CaBPs at synapses providing inputs to low- or high-threshold SGN fibers. Apart from a small shift in voltage activation and abundance of the channels ⁴⁸, not much is known about the possible differences in the Ca_V1.3 properties and interaction partners at distinct ribbons. Neither is it known whether the composition of the channel complex [t.i. proportions of alpha subunit splice variants ^19,49,50, and different beta subunits ⁵¹] differs systematically between ribbon-AZs partnered by different classes of SGN fibers. Investigation of intracellular Ca²⁺ signals from individual AZs may offer some answers in the future. Different amounts of rescue of threshold vs suprathreshold responses could be further due to confounding effects of CaBP1 and 2, e.g. augmented SGN response in Cabp2-injected DKO animals still lacking CaBP1 (reminiscent of the phenotype of young Cabp1 KOs) in combination with “only” partial rescue of CaBP2 function. Possible adverse effects of exogenous expression of CaBP2, e.g. off-target toxicity and uncontrolled overexpression of the transgene, have been discussed previously ²¹, and further experiments need to be performed in the future to achieve (close to) complete rescue of hearing function in these mutants.

Our results reveal that the amount of Ca_V1.3 inactivation increases dramatically when IHCs lack both major CaBPs. This suggests that the sole loss of either CaBP1 or 2, can be largely compensated by the other of the two CaBP members. Yet, the loss of CaBP2 alone still results in progressive hearing impairment in mice and DFNB93 in humans. The inability of CaBP1 to fully replace CaBP2 in the CaBP2-deficient IHCs may stem from either too low concentration of CaBP1 (and other CaBPs), or from distinct properties of the two proteins (enabling CaBP2 to exert more potent suppression of Ca_V1.3 inactivation, e.g. VDI, or potentially performing additional, yet unidentified cellular function). Interestingly, even the overexpression of CaBP2 did not completely rescue the hearing phenotype of DKO animals. Future experiments involving overexpression of CaBP1 (and possibly CaBP1-2 molecular chimeras) in Cabp2 SKO and/or Cabp1/2 DKO animals together with further biophysical interaction studies are required to gain a better understanding of the potentially distinct functions performed by the two major CaBPs in the hair cells.

CaBPs as the regulators of IHC synaptic transmission

Previously, patch-clamp recordings surprisingly revealed normal whole-cell exocytosis in CaBP2-deficient IHCs despite enhanced inactivation of calcium channels ¹³, and no obvious defects in the CaBP1-deficient IHCs ¹². In contrast, IHCs deficient in both, CaBP1 and 2, displayed strongly impaired exocytosis. Transgenic overexpression of CaBP2 in IHCs resulted in a partial recovery of the Ca²⁺ influx and an even better recovery of exocytosis. Together, these results corroborate the hypothesis of the role of CaBPs in preventing extensive steady-state inactivation of Ca_V1.3 channels to support sufficient synaptic exocytosis for proper sound encoding ¹³. The lack of CaBP1 and 2 seems to impair sustained exocytosis and efficiency of exocytosis beyond the levels that could be attributed to reduced Q_Ca. This became particularly apparent when the IHCs were challenged by higher holding potentials in combination with large sine wave amplitudes during capacitance measurements, which we assume to partially deplete the pool of RRP vesicles. At present, it is not clear how CaBPs may directly affect the exocytosis, e.g. by influencing the coupling of the Ca_V1.3 channels and synaptic vesicles or potentially affecting the expression levels of other synaptic proteins that mediate synaptic vesicle replenishment. In this respect, it is worth noting that CaBP1 and 2 have been reported to modulate CaMKII activity ³, which in turn may through phosphorylation modify the Ca²⁺ affinity of otoferlin C2 domains ⁵². In the future, it is thus essential to investigate if CaBPs regulate the function of otoferlin and thus synaptic processes. Furthermore, other members of the CaBP/CaM family have been suggested to interact with synaptic proteins and/or be involved in the regulation of synaptic vesicle cycle: CaM in endocytosis ⁵³, CaBP5 in priming via interaction with Munc18-1 ⁵⁴, CaBP1 via inhibition of IP₃ receptors ^55,56. An important challenge for future studies is to identify further CaBP(2)-interaction partners and possible functions beyond modulation of calcium channels.

The recovery of Ca_V1.3 channels from inactivation upon prolonged milder stimulation (2 min sine wave) was following a single exponential course in WT IHCs, but additional slower component was detected in the IHCs deficient in CaBP1 and 2. In the IHCs of injected DKO animals, this second component appeared smaller and faster, suggesting that CaBPs speed-up recovery of Ca_V1.3 channels from inactivation. Upon a comparably shorter (500 ms) but stronger depolarization, the recovery of Ca_V1.3 from inactivation was very fast in DKO IHCs, in fact faster than in (WT or) Cabp2-injected DKO IHCs. We hypothesize this to be due to faster Ca²⁺ clearance from the DKO IHC synapses as the intracellular [Ca²⁺] levels are expected to rise significantly less in CaBP1/2-deficient IHCs due to fast and pronounced Ca_V1.3 inactivation. In contrast, higher [Ca²⁺] may build- up at the synapses of WT and “rescued” DKO IHCs; consequently, CDI may be prolonged. Another plausible explanation for the observation is, if we consider that CaPB2 may act as a slow mobile calcium buffer ¹³. In the presence of excessive “overexpressed” CaBP2 in injected IHCs of DKO animals, the depolarization-evoked intracellular [Ca²⁺] transients may become biexponential as has been described for parvalbumin alpha ⁵⁷, which may prolong the time constant of Ca²⁺ clearance and determine the kinetics of channel recovery from inactivation. Our results of the recovery upon 500-ms depolarization may thus mostly reflect the kinetics of the Ca²⁺ clearance from the cytosol, while the experiments with prolonged sine wave stimulation (when e.g. the function of calcium pumps and other extrusion systems and intracellular [Ca²⁺] levels may be at equilibrium), may report the speed of calcium channel recovery from a slow inactivation. Slow inactivation with very slow recovery was described for Na_V, K_V and Ca_V channels ^27–30. It has to be noted that the time constant of recovery of Ca_V1.2 from slow VDI was comparably shorter (4 s) as observed in the present study ³⁰. Possibly different conditions (e.g. stimulus parameters) or channel subtype may underlie this difference. Future experiments combining patch-clamp recordings with calcium imaging should be employed to address this question further. Finally, while we consider the effects of surgery on Ca_V1.3 recovery kinetics in injected mice as unlikely, we can at present not exclude this possibility.

From our in vitro recordings at higher holding potential or upon prolonged stimulation with small voltage oscillations we predict that in vivo a noteworthy proportion of Ca_V1.3 channels may be effectively inactivated and not available for mediating IHC synaptic transmission. This could explain the low experimental yield, the low “spontaneous” spiking rates of the SGNs and lower sound-evoked spike rates with higher thresholds. Similarly, thresholds may be high for tone bursts in the ABR measurements due to high proportion of inactivated channels upon stimuli at high repetition rate used for these recordings (20-40 Hz repetition rate for click and tone burst stimulation, respectively). We believe that any sound stimulation and potentially even spontaneous activity may effectively silence DKO synapses. As expected, longer interstimulus intervals may allow a larger pro-portion of channels to recover between stimuli, increasing the onset rates of SGN spiking while decreasing the first spike latency and jitter. Still, high-intensity stimulation had to be used in the DKO animals to evoke an increase in SGN spiking. It is possible that spontaneous activity and/or our routines used to determine the SGN characteristic frequency inactivate a majority of IHC Ca_V1.3 channels thus not allowing reliable assessment of the characteristic frequency. In addition to Ca_V1.3 channel inactivation, other factors may contribute to low hearing sensitivity, such as possible impairment of synaptic vesicle replenishment, or IHC mechanotransduction, and, in some cases, impaired cochlear amplification. A complete lack of BK channels was shown to affect the IHC resting potential, receptor potential amplitude and timing, and consequently the timing and frequency of spiking in the SGNs ⁵⁸. Lower expression of BK channels is not expected to significantly affect hearing thresholds, at least initially ^59–61, and thus likely does not contribute to the hearing defect of the Cabp1/2 DKO animals. However, at advanced ages, the effects described above may combine with the lack of CaBPs to additionally desynchronize already scarce spikes in the auditory nerve fibers.

Clinical implications for the diagnosis and treatment of DFNB93

The SGN sound encoding phenotype in Cabp2-KO mice was surprisingly mild compared to the deficits observed in DFNB93 patients ^{e.g. 15,13}: SGN thresholds and evoked spike rates were only minimally affected ¹³. Given the strong recovery phenotype in the DKO we now consider the possibility that synaptic activity in response to everyday sounds could already inactivate calcium channels and reduce SGN spiking. In this case, communication in daily life would be more strongly impaired than audiometric data obtained in a soundproof chamber, especially when short stimuli separated by silent intervals are used. In mild cases, a gap detection test may be used to assess auditory fatigue. Based on our data we further predict that patients that have a stronger channel inactivation phenotype might not benefit much from sound amplification by hearing aids. Instead, they should use technologies that improve the signal to noise ratio (e.g. noise cancellation, directional microphones, wireless transmitters). An alternative or additional future treatment approach may be gene therapy. In our hands, re-expression of CaBP2 by genetic treatment partially restored Ca_V1.3 properties, IHC exocytosis and consequently ABR wave amplitudes and thresholds. This successful viral rescue supports the view that a reversible inactivation of IHC Ca_V1.3 currents is the main mechanism of hearing loss in CaBP-related deafness, which can be targeted by gene therapy in the future.

Materials and Methods

Animals

For the generation of the Cabp1/Cabp2-double-knockout (Cabp1/2-DKO) line, CaBP2^LacZ/LacZ mice, carrying LacZ trapping cassette in place of the exons 3 and 4 ¹³, were crossbred with Cabp1 KO mice ⁶² and maintained on a C57/Bl6 background. Mice from 3-13 weeks were used for electro-physiological and systems physiology recordings. All experiments complied with the national animal care guidelines and were approved by the University of Göttingen Board for animal welfare and the animal welfare office of the state of Lower Saxony.

Systems physiology recordings: ABR and DPOAE

Prior to the experiments mice were weighed and anesthetized via intraperitoneal injections of ketamine (125 µg/g) and xylazine (2.5 µg/g). Recordings were obtained in a custom-made soundproof box with a Tucker-Davis (TDT) BioSig System II/BioSig software (Tucker-Davis Technologies, Alachua, FL). Body temperature was maintained using a heating pad (37°C) and ECG was monitored continuously. For ABR recordings, subcutaneous needles were placed at the vertex, the ipsilateral pinna and at the lower back. Using a JBL2402 speaker in free-field configuration, tone bursts (10-ms plateau, 1-ms cos² rise/fall) or clicks of 0.03 ms were ipsilaterally presented at 40 Hz for tone bursts or 20 Hz for clicks. ABR traces were amplified 50000-fold, band-pass filtered between 400 Hz and 4 kHz (NeuroAmp) and averaged from 1300 repetitions sampled at 50 kHz for 20ms. Thresholds were defined in stacked waveforms as the lowest stimulus intensity (dB SPL) at which a reproducible waveform could be visually detected with a 10 dB precision. Wave I amplitudes were measured peak-to-trough. DPOAEs were measured using a custom-written MATLAB (MathWorks) routine. Two primary tones f1 and f2 (ratio f1/f2 = 1.2, level difference f2 = f1 + 10 dB) were presented via two speakers (MF-1, TDT) and a custom-made ear probe containing an MKE-2 microphone (Sennheiser). Stimulus duration was 16 ms and f2 levels varied from 10 to 70 dB SPL in 10 dB steps. The microphone signal was amplified (DMX 6Fire; Terratec (young animals), or UAC-2; Zoom (older animals)) and digitized (TDT System III). DPOAE amplitude was analyzed at 2×f2-f1 using custom-written MATLAB software (MathWorks) with fast Fourier transformation. DPOAE thresholds were determined as responses that exceeded -18 dB SPL in young and -4 dB SPL in older animals.

Auditory nerve fiber recordings

Mice were anesthetized by injection of xylazine (5 mg/kg), urethane (1.32 mg/kg) and buprenorphine (0.1 mg/kg) i.p., tracheotomized, and placed in a stereotactic apparatus. The left occipital bone and the cerebellum were partially removed to visualize the left superior semicircular canal. The latter served as a landmark to navigate a glass microelectrode through the cochlear nucleus, aiming at the location where the auditory nerve leaves the internal auditory canal and enters the cochlear nucleus. 85 or 90 dB broadband noise bursts (ScanSpeak, Avisoft Bioacoustics) were used as search stimuli to identify neurons that displayed sound-driven spiking activity. They were characterized by spontaneous rate and frequency tuning and the responses to tone bursts at the characteristic frequency, 30 dB above the threshold, as previously described ³⁶. In the majority of sound-responsive DKO neurons, thresholds were so high and tuning so broad that neither the automated tuning curve algorithm nor systematic or manual changes in sound frequency elicited frequency-specific responses. In these cases, we either chose a subjective “best frequency” or used white noise burst stimulation at 85 dB.

Patch-clamp recordings

For patch-clamp recordings, the apical coils of 3-4-week-old animals were dissected in HEPES-Hanks solution containing (in mM): 10 HEPES, 5.35 KCl, 141.7 NaCl, 0.5 MgSO₄, 1 MgCl₂, 11.1 D-glucose and 3.42 L-glutamine. pH was adjusted to 7.2. IHCs of the ∼6 kHz tonotopical region were investigated using the perforated patch configuration. The pipette solution contained (in mM): 130 Cs-gluconate, 10 TEA-Cl, 10 4-AP, 1 MgCl₂, 10 HEPES, 300 mg/mL amphotericin B, pH 7.2 ∼290 mOsm. The bath solution was composed of (in mM): 111 NaCl, 35 TEA-Cl, 2.8 KCl, 1 CsCl, 1 MgCl₂, 10 NaOH-Hepes, 11.3 D-glucose, and 1.3 or 2 CaCl₂ or BaCl₂ (pH 7.2, 300–310 mOsm). When the bath solution contained 2 mM BaCl₂/CaCl₂, NaCl was adjusted to 110 mM. The bath solution was supplemented by the SK2 channel blocker apamin (100 nM; Peptanova, Germany) freshly on the day of the experiment. Recordings were performed at room temperature using an EPC-9 amplifier (Heka-Germany) controlled by Pulse software. Currents were leak-corrected using a p/10 protocol, sampled at 50 kHz and corrected offline for the liquid junction potential (-15 mV).

IHCs were patched at a holding potential of -85 mV (in a subset of experiments, -65 and -55 mV) and stimulated with depolarizations to the peak Ca²⁺-current potential. Changes in membrane capacitance were monitored by applying a 2-kHz-sinewave with an amplitude of 35 mV. In a subset of experiments, the amplitude was reduced to 15 mV to diminish possible stimulation of the cells caused by the sinewave. VDI and CDI were calculated as follows: VDI = 1 - I_{Ba_500}/I_{Ba_initial} and CDI = 1 − (I_{Ca_500}/I _{Ca_initial})/(I_{Ba_500}/I_{Ba_initial}), where I_{Ba_500} and I_{Ca_500} represent whole cell Ba²⁺ and Ca²⁺ currents 500 ms after beginning of depolarization, and I_{Ba_initial} and I_{Ca_initial} correspond to the initial, peak currents with the respective divalent cations ²³.

Immunohistochemistry

Apical coils (6-8 kHz tonotopical region) of the organ of Corti were fixed with 4% (vol/vol) formaldehyde in PBS on ice. After incubation for 1 h at room temperature in goat serum dilution buffer [16% (vol/vol) normal goat serum, 450 mM NaCl, 0.6% Triton X-100, 20 mM phosphate buffer (pH 7.4)], the explants were incubated in primary antibodies over night at 4 °C. The following antibodies were used: chicken anti-Homer1 (1:200, Synaptic Systems), rabbit anti-Myosin6 (1:200, Proteus), guinea pig anti-Synapsin1/2 (1:200, Synaptic Systems), rabbit anti-RibeyeA (1:200, Synaptic Systems), guinea pig anti–parvalbumin-α (1:200; Synaptic Systems), rabbit anti-BK (1:100; Alomone), rabbit anti-SK2 (1:200; Alomone), mouse IgG1 anti-CtBP2 (1:200; BD Biosciences), rabbit anti-GluA3 (1:200; Santa Cruz), rabbit–anti-CaV1.3 (1:100; Alomone), chicken anti-GFP (1:200, Abcam). The explants were then incubated with secondary AlexaFluor-labeled antibodies (1:200; Molecular Probes) for 1 h at room temperature, washed 3 times in PBS and mounted. Confocal images were acquired with an Abberrior Instruments Expert Line STED microscope using a 1.4 NA 100x oil immersion objective. ImageJ software (NIH) was employed to analyze 2D confocal stacks and generate maximum projections. Volumetric synapse and BK-cluster immunofluorescence analysis ran on Imaris (Bitplane, Zurich, Switzerland) paired with a custom-written Matlab software ⁶³.

Molecular biology and AAV vector production

The construct containing the long isoform of mouse CaBP2 (CaBP2-L; NCBI Reference Sequence: NP_038906.2) and eGFP is depicted in Fig. S1A. AAV9-PHP.eB particles were generated using our standard AAV purification procedure previously described in more detail in ref. ⁶⁴. In brief, triple transfection of HEK-293T cells was performed using pHelper plasmid (TaKaRa/Clontech), the trans-plasmid providing viral capsid PHP.eB (a generous gift from Viviana Gradinaru, Addgene plasmid #103005) and the cis-plasmid providing hCMV/HBA_wtCaBP2-P2A-eGFP. PHP.eB viral particles were harvested from the medium 72 h after, and from cells and the medium 120 h after transfection. Precipitation was done with 40% polyethylene glycol 8000 (Acros Organics, Germany) in 500 mM NaCl for 2 h at 4°C. Both, precipitate and cells were lysed in high salt buffer (500 mM NaCl, 2 mM MgCl₂, 40 mM Tris-HCl, pH 8,0) and non-viral DNA was degraded using salt-activated nuclease (SAN, Arcticzymes, USA). The cell lysates were clarified by centrifugation at 2000×g for 10 min and then purified over iodixanol (Optiprep, Axis Shield, Norway) step gradients (15, 25, 40, and 60%) at 350000×g for 2.25 h ^65,66. Finally, viral particles were concentrated using Amicon filters (EMD, UFC910024) and formulated in sterile PBS supplemented with 0.001% Pluronic F-68 (Gibco, Germany). Virus titer was 8.2×10¹² genome copies/ml measured by determining the number of DNase I resistant genome copies using qPCR (StepOne, Applied Biosystems) and AAV titration kit (TaKaRa/Clontech). The purity of viruses was routinely checked by silver staining (Pierce, Germany) after gel electrophoresis (NovexTM 4-12% Tris-Glycine, Thermo Fisher Scientific).

Virus injections

Mice were injected at postnatal day 6 using the round window approach as described in earlier studies ⁶⁷. In brief, anesthesia was established with isoflurane (5% for induction, 2-3% for maintenance, frequent testing of the absence of hind-limb withdrawal reflex). For analgesia, buprenorphine (0.1 mg/kg, 30 min before surgery) and carprofen (5 mg/kg, during and 1 day after surgery) were applied subcutaneously and Xylocain (10 mg spray) locally. Body temperature was maintained by a custom-built heating blanket. Following a retro-auricular approach, the facial nerve was exposed to determine where to puncture the cartilaginous bulla with the injection pipette and target the scala tympani where virus suspension (1-1.5 μl, corresponding to 0.82-1.23×10¹⁰ AAV particles) was injected. Following injection, the endogenous tissue was relocated and the surgical situs closed by suturing the skin. 2-3 weeks later, the injected mice were used for hearing tests, in vitro electrophysiology and immunohistochemistry of the cochleae.

Data Analysis

Data were analyzed using Igor Pro Software (Wavemetrics), GraphPad PRISM (Graphpad Software, Inc.), Matlab (Mathworks) and Imaris (Bitplane). All data are presented as mean ± SEM. For the analysis of half-max Ca²⁺ current (V_half) and activation constant (k_act), current-voltage traces were transformed into an activation function (I_Ca = g_max(V-V_rev)), which was subsequently fitted by a Boltzmann function: 1/(1 + exp((V_0.5−V)/k_act)). G_max describes the maximum conductance, V the holding potential and V_rev the reversal potential. Data were tested for randomness, normality (Jaque-Bera test), equality of variances (F-test) and finally compared for statistical significance by performing Student’s t-test for normally distributed data of equal variance or else by the Wilcoxon rank sum test. Systems physiology data were tested for significance using ANOVA with Tukey’s or Šidak’s multicomparisons tests.

Acknowledgements

The authors would like to thank Christiane Senger-Freitag for the virus injections, Daniela Gerke for help with virus production, and Sandra Gerke, Ina Preuss, and Sina Langer for excellent technical support. The authors would further like to thank Amy Lee (University of Iowa) for providing the Cabp1 KO animals, and Mohona Mukhopadhyay and Tobias Moser for critical reading of the paper, useful comments and suggestions. This work was supported by the German Research Foundation: DFG Priority Program 1608 “Ultrafast and temporally precise information processing: Normal and dysfunctional hearing” [to TP (PA 2769/1-1)], the Clinician Scientist Program “Cell Dynamics in Disease and Therapy” at the University Medical Center Göttingen (to DO; project number 413501650), CRC889 (subproject A06; to NS, and B09; to TP); and by the Multiscale Bioimaging Cluster of Excellence (EXC 2067/1-390729940; to TP).