Current Response in CaV1.3–/– Mouse Vestibular and Cochlear Hair Cells

Signal transmission by sensory auditory and vestibular hair cells relies upon Ca2+-dependent exocytosis of glutamate. The Ca2+ current in mammalian inner ear hair cells is predominantly carried through CaV1.3 voltage-gated Ca2+ channels. Despite this, CaV1.3 deficient mice (CaV1.3–/–) are deaf but do not show any obvious vestibular phenotype. Here, we compared the Ca2+ current (ICa) in auditory and vestibular hair cells from wild-type and CaV1.3–/– mice, to assess whether differences in the size of the residual ICa could explain, at least in part, the two phenotypes. Using 5 mM extracellular Ca2+ and near-body temperature conditions, we investigated the cochlear primary sensory receptors inner hair cells (IHCs) and both type I and type II hair cells of the semicircular canals. We found that the residual ICa in both auditory and vestibular hair cells from CaV1.3–/– mice was less than 20% (12–19%, depending on the hair cell type and age investigated) compared to controls, indicating a comparable expression of CaV1.3 Ca2+ channels in both sensory organs. We also showed that, different from IHCs, type I and type II hair cells from CaV1.3–/– mice were able to acquire the adult-like K+ current profile in their basolateral membrane. Intercellular K+ accumulation was still present in CaV1.3–/– mice during IK,L activation, suggesting that the K+-based, non-exocytotic, afferent transmission is still functional in these mice. This non-vesicular mechanism might contribute to the apparent normal vestibular functions in CaV1.3–/– mice.


INTRODUCTION
The inner ear houses the auditory and the balance organs. In mammals, the primary sensory cells are the inner hair cells (IHCs) of the cochlea, and the type I and type II hair cells of the vestibular system. Acoustic stimuli or head movements cause change in the hair cell membrane potential, which modulates Ca 2+ inflow and related neurotransmitter (glutamate) exocytosis (Bonsacquet et al., 2006;Dulon et al., 2009;Songer and Eatock, 2013;Sadeghi et al., 2014;Vincent et al., 2014;Kirk et al., 2017). Both auditory and vestibular hair cells express voltage-gated L-type Ca 2+ channels containing the pore-forming Ca V 1.3 subunit (previously known as α1D), which are characterized by a negative voltage of activation (about −60 mV) and negligible voltage-dependent inactivation (Platzer et al., 2000;Bao et al., 2003;Johnson and Marcotti, 2008;Zampini et al., 2013). Pharmacologically, L-type (Ca V 1.1 to Ca V 1.4) Ca 2+ channels are identified by their sensitivity to dihydropyridines (DHPs) such as nimodipine and nifedipine (antagonists) or BayK 8644 (agonist), which do not affect the other voltage-gated Ca 2+ channels (Ca V 2 and Ca V 3). However, the Ca V 1.3 subunit is relatively insensitive to DHP antagonists compared to Ca V 1.1, 1.2, and 1.4 subunits (Koschak et al., 2001;Xu and Lipscombe, 2001).
The majority (∼90%) of the Ca 2+ current in IHCs is carried by the Ca V 1.3 subunit (Platzer et al., 2000;Jeng et al., 2020a). The nature of the remaining ∼10% of the Ca 2+ current is still unknown (see Pangrsic et al., 2018), although previous work has indicated that it could be carried by the Ca V 1.4 subunit (Brandt et al., 2003). Consistent with the critical role of Ca V 1.3 channels in hair cell Ca 2+ dependent exocytosis, Ca V 1.3 −/− mice are deaf (Platzer et al., 2000;Brandt et al., 2003), but do not show vestibular deficits (Platzer et al., 2000;Dou et al., 2004). A pharmacological study from rat semicircular canal crista hair cells has indicated that the level of expression of Ca V 1.3 Ca 2+ channels is comparable to that of the cochlear IHCs (Bao et al., 2003). However, vestibular utricle hair cells from Ca V 1.3 −/− mice appear to express a large residual Ca 2+ current (∼50%, Dou et al., 2004), which could potentially drive some signal transmission to the afferent fibers, at least during linear horizontal head accelerations. Currently, it is unknown whether a substantial residual Ca 2+ current is also expressed in Ca V 1.3 −/− semicircular canal hair cells, which could compensate for rotation-related reflexes (e.g., vestibulo-ocular reflexes), and whether its size is similar between type I and type II hair cells. The possible presence of large residual Ca 2+ currents in vestibular hair cells from Ca V 1.3 −/− mice, together with the recently identified non-quantal, Ca 2+ -independent, signal transmission at type I hair cell synapses (Eatock, 2018) could contribute, at least in part, to the milder vestibular phenotype present in Ca V 1.3 −/− mice. Non-quantal transmission, which has not been reported in auditory hair cells, involves intercellular K + accumulation in the synaptic cleft occurring during the activation of a hyperpolarizing-activated outward rectifying K + current I K,L (Lim et al., 2011;Contini et al., 2012Contini et al., , 2017Contini et al., , 2020Spaiardi et al., 2017Spaiardi et al., , 2020a. Here, we have performed whole-cell patch clamp recordings from IHCs and both type I and type II hair cells from the mammalian crista of Ca V 1.3 −/− mice using the same experimental conditions in terms of extracellular Ca 2+ and temperature. We have found that both IHCs and vestibular hair cells from Ca V 1.3 −/− mice have < 20% of residual Ca 2+ current compared to control mice. Thus, differences in Ca V 1.3 Ca 2+ channel expression among vestibular organs (saccule vs. semicircular canals) may exist, possibly allowing hair cells from some vestibular organs to retain some Ca 2+ -dependent neurotransmitter exocytosis. We have also found that, different from cochlear IHCs, Ca V 1.3 Ca 2+ channels are not required for the normal maturation of the biophysical properties (K + channel expression) of both type I and type II hair cells. Since I K,L and intercellular K + accumulation in the synaptic cleft of type I hair cells were normal in Ca V 1.3 −/− mice, non-quantal signal transmission might also contribute to the vestibular functioning of Ca V 1.3 −/− mice.

Ethics Statement
All animal work was performed at the University of Sheffield (United Kingdom), licensed by the Home Office under the Animals (Scientific Procedures) Act 1986 (PPL_PCC8E5E93) and approved by the University of Sheffield Ethical Review Committee (180626_Mar). For all in vitro work, mice were culled by cervical dislocation, which is a schedule 1 method.

Tissue Preparation
Experiments were performed using Ca V 1.3 knockout mice (Ca V 1.3 −/− ) on a C57BL/6N background and control mice (littermate heterozygous or C57BL/6N). Mice from both sexes were used and ranging from postnatal day 4 (P4) to P35. The semicircular canals with their ampullae and cochleae (apical turn) were dissected out from the inner ear as reported previously (Jeng et al., 2020aSpaiardi et al., 2020a;Carlton et al., 2021), using an extracellular solution composed of (in mM) 135 NaCl, 5.8 KCl, 1.3 CaCl 2 , 0.9 MgCl 2 , 0.7 NaH 2 PO 4 , 5.6 Dglucose, and 10 HEPES-NaOH. Sodium pyruvate (2 mM), amino acids, and vitamins were added from concentrates (Thermo Fisher Scientific, United Kingdom). The pH was adjusted to 7.5 (osmolality ∼308 mmol kg −1 ). The dissected organs were fixed at the bottom of the recording chamber by a nylon-meshed silver ring and were continuously perfused with the above extracellular solution (0.5 ml/min) using a peristaltic pump (Masterflex L/S, Cole Palmer, United States). Hair cells were viewed using a upright microscopes (Olympus BX51; Leica DM-LFS) equipped with Nomarski Differential Interface Contrast (DIC) optics with a 60X or 64X water immersion objective and x15 eyepieces.

Whole-Cell Electrophysiology
Voltage-clamp whole-cell experiments were performed at room temperature (18-22 • C) for K + current recordings, and nearbody temperature (32-36 • C) for Ca 2+ currents recordings using an Optopatch amplifier (Cairn Research Ltd., United Kingdom) as previously described (Johnson and Marcotti, 2008;Jeng et al., 2020b;Spaiardi et al., 2020b). The patch pipettes were pulled to 2-3 M tip resistance from soda glass capillaries (Hilgenberg, Germany) and coated with surf-wax (Mr. Zoggs SexWax, United States) to minimize the fast capacitance transient across the wall of the patch pipette. For K + current recordings, the patch pipette filling solution contained (in mM): KCl 131, Na 2 -Phosphocreatine 10, MgCl 2 3, EGTA-KOH 1, Na 2 ATP 5, and HEPES 5; pH adjusted to 7.28 with KOH (osmolality was 294 mmol kg −1 ). For Ca 2+ current recordings, the pipette intracellular solution contained (in mM): 106 Cs-glutamate, 20 CsCl, 3 MgCl 2 , 1 EGTA-CsOH, 5 Na 2 ATP, 0.3 Na 2 GTP, 5 HEPES-CsOH, 10 Na 2 -phosphocreatine, pH 7.3 with CsOH (294 mmol kg −1 ). Data acquisition was controlled by pClamp software using a Digidata board (Molecular Devices, United States). Recordings were low-pass filtered at either 2.5 or 5 kHz (8-pole Bessel), sampled at 5, 10, or 100 kHz and stored on computer for offline analysis using Clampfit (Molecular Devices, United States) and Origin (OriginLab, United States) software. Membrane potentials reported in the text and figures were corrected for the uncompensated residual series resistance (R s ) and the liquid junction potential (LJP), which was either −4 mV for the K +based and −11 mV for the Cs + -glutamate-based intracellular solution, measured between electrode and bath solutions. For K + current recordings from vestibular hair cells, R s was calculated off-line from the capacitive artifact elicited by applying a voltage step from either −74 to −64 mV in type II and −124 to −44 mV in type I hair cells. The different voltage step was used in order to minimize artifact contamination by inward and outward rectifier voltage-gated channels in the two hair cell types (Spaiardi et al., 2017). Voltage clamp protocols are referred to a holding potential of −64 mV for the K + -based intracellular solution or −91 mV for the Cs + -based intracellular solution.

Statistics Analysis
Statistical comparisons of means were made by Student's twotailed t-test or, for multiple comparisons, analysis of variance (one-way or two-way ANOVA) was applied. P < 0.05 was selected as the criterion for statistical significance. Mean values are quoted as means ± SD.

Potassium Currents Recorded From
Type I and Type II Crista Hair Cells From Control and Ca V 1.3 −/− Mice Type I hair cells of the ampullae sensory epithelium (the crista) exhibit a large low-voltage activated outward rectifying K + currents, named I K,L (Rennie and Correia, 1994;Rüsch and Eatock, 1996), which was recorded in both adult wild-type ( Figure 1A) and Ca V 1.3 −/− mice ( Figure 1B). Since I K,L is almost completely activated at −60 mV, hyperpolarizing voltages from the holding potential of −64 mV produced deactivating tail currents (e.g., −104 and −124 mV: Figures 1A,B), while depolarizations elicit an instantaneous increase of the outward currents. The mean steady-state current-voltage (I-V) relationship for the total current recorded in type I hair cells was not significantly different between wild-type (P17-P22; n = 12) and Ca V 1.3 −/− mice (P19-P20; n = 6) (P = 0.9742, F = 0.4149, DFn = 15, two-way ANOVA, Figure 1C). The negligible steadystate current at −124 mV is consistent with I K,L being fully deactivated at this potential, and with the absence of inward rectifier currents in mouse crista type I hair cells (Spaiardi et al., 2020a). Note that the outward tail currents in type I hair cells, which were recorded at −44 mV, were smaller following the voltage step to −24 mV than those at −44 mV in both wildtype (Figures 1A,D) and Ca V 1.3 −/− mice (Figures 1B,E,F). The presence of progressively smaller outward tail current following larger outward K + currents (Figures 1D,E), which in some cases includes tail current reversal (Figure 1F), is consistent with intercellular (e.g., in the calyceal synaptic cleft) K + accumulation inducing a shift in the K + equilibrium potential (Lim et al., 2011;Contini et al., 2012Contini et al., , 2017Contini et al., , 2020Spaiardi et al., 2020a). Progressive intercellular K + accumulation during depolarization is also responsible for the "apparent" inactivation of the outward current, which is due to the progressive decrease of the driving force for K + to exit the hair cells (e.g., Spaiardi et al., 2017).
Different from the type I hair cells, the macroscopic current recorded from adult type II hair cells is not dominated by I K,L (Figures 1G,H), but instead express several other currents that have been previously described in great details (Meredith and Rennie, 2016). This include a transient (I K,A ) and a delayed rectifying (I K,v ) outward K + current, an inward rectifying K + current (I K,1 ), and the mixed inward rectifying Na + /K + current (I h ). All these currents were evident from the inward and outward current profile recorded from type II hair cells of Ca V 1.3 −/− mice ( Figure 1H). The mean steady-state I-V for the total current recorded in type II hair cells was also not significantly different between wild-type (P17-P22; n = 4) and Ca V 1.3 −/− mice (P18-P19; n = 4) (P = 0.7010, F = 0.7756, DFn = 15, two-way ANOVA, Figure 1I).
The above results demonstrate that the absence of the Ca V 1.3 Ca 2+ channel subunit does not impair the normal developmental acquisition of voltage-dependent K + currents in vestibular type I and II hair cells in the crista. This is different from the cochlea, where the normal expression of the K + currents characteristic of adult inner hair cells (IHCs), which are the fast activating BK current I K,f and negatively activating delayed rectifier I K,n (Kros et al., 1998;Marcotti et al., 2003;Oliver et al., 2003), was either prevented (apical-coil) or reduced (basal-coil) in Ca V 1.3 −/− mice (Brandt et al., 2003;Jeng et al., 2020a).
Calcium Currents in Type I and Type II Hair Cells in Control and Ca V 1.3 −/− Mice Although type I and type II hair cells are morphologically distinct, this is not always visible when working with the intact organ. Therefore, the correct identification of type I hair cells relies on the presence of I K,L (Figure 1). Recordings were performed using intracellular Cs + , which is known to block I h (Biel et al., 2009) and the outward rectifying K + currents (Bao et al., 2003) in type II hair cells while having little effect on I K,L (Griguer et al., 1993;Rüsch and Eatock, 1996;Chen and Eatock, 2000;Rennie and Correia, 2000). An additional consideration affecting the correct identification of I K,L is its state of deactivation at the holding potential used for the recordings, which is generally set at −90 mV. Figures 2A,C shows representative current responses recorded from a type I hair cell with a largely deactivated I K,L , as indicated by the small inward current at −91 mV, and the small instantaneous current upon depolarizing and hyperpolarizing voltage steps. In the example shown in Figures 2B,C, however, I K,L is largely activated at −91 mV and exhibits large instantaneous currents. Although this variability has previously been reported (Hurley et al., 2006;Spaiardi et al., 2017), we found that the reversal potential for the macroscopic current was significantly more hyperpolarised in type I hair cells showing a largely activated I K,L at −91 mV (Figure 2C). While a deactivated I K,L at the holding potential was primarily recorded in early postnatal hair cells (n = 15), more mature cells tend to exhibit a largely activated current (n = 8) ( Figure 2D). Since the reversal potential of the macroscopic K + that was (red), or was not (black), completely deactivated at −111 mV. Each symbol refers to the steady-state value of I K,L measured in each cell at −111 mV. Right: same data as in the left panel but subdivided into type I hair cells that are completely deactivated (red) and not deactivated (black) at −111 mV clusters using k-means clustering. Arrows indicate the hair cells that are located in one cluster but have I K,L properties that fit best the other cluster. current in type I hair cells is primarily determined by the ions flowing through the K,L channels, it should reach values near the reversal potential of the mixed Cs + /K + current (called a "biionic potential"; see Hille, 2001) through K,L channels in the presence of a large I K,L and intracellular Cs + (Spaiardi et al., 2020a). Given the reported permeability ratio of Cs + to K + of about 0.31 (Rüsch and Eatock, 1996;Spaiardi et al., 2020a), the reversal potential should be close to −40 mV. The finding that the reversal potential in older hair cells ( Figure 2C) is close to −60 mV could indicate that the relative permeability of K,L channels to Cs + is likely to be larger than that previously estimated, at least in more mature type I hair cells. However, the presence of a residual calyx might also produce a shift of the mixed Cs + /K + equilibrium potential toward more negative voltages during inward currents (Spaiardi et al., 2017(Spaiardi et al., , 2020a. This would imply a tighter attachment of the calyx to more mature type I hair cells. The above results are consistent with I K,L increasing in amplitude and activating at more hyperpolarized potentials during post-natal development, as also previously shown in rat type I hair cells (Hurley et al., 2006). Different K + channels subunits have also been found to be expressed by rodent type I hair cells during postnatal development which may account for the above changes (Hurley et al., 2006;Spitzmaul et al., 2013).
Following the positive identification of the patched hair cell as type I, a small inward current (Figure 3A, upper panel) became visible when superfusing the cells with an extracellular solution containing the K + channel blockers TEA and 4AP, in the presence of 5 mM Ca 2+ (see section "Materials and Methods"). In a subset of type I hair cells (n = 8), we found that the addition of 0.1 mM Cd 2+ to the extracellular solution fully blocked the inward current, confirming its identity as I Ca (Figure 3A, lower panel). The isolated I Ca was obtained by subtracting the current recorded in the presence of TEA, 4AP, and Cd 2+ to that obtained in the absence of Cd 2+ (Figure 3B). Under this experimental condition, the inward current activated at about −61 mV and peaked near −21 mV (Figures 3B,D). Comparable results were obtained when I Ca was isolated by performing the leakage-subtraction procedure (see section "Materials and Methods") to the currents recorded in TEA and 4AP (Figures 3C,D).  In Ca V 1.3 −/− mice, the inward current was barely visible in type I hair cells when the extracellular solution contained TEA, 4AP, and 5 mM Ca 2+ (Figure 3E). Following the isolation of I Ca with Cd 2+ (Figure 3F), or after leakage subtraction (Figure 3G), it became more easily detectable. The inward current peaked near −11 mV ( Figure 3H). In type I hair cells, the mean peak I Ca after Cd 2+ -subtraction was 121 pA in wild-type and 19 pA in Ca V 1.3 −/− mice (15.7% of the wilt-type value).
For type II hair cells, the isolation of the Ca 2+ current was performed as shown in Figure 3, using either the isolated Cd 2+ -procedure (Figures 4A,D) or after leakage subtraction (Figures 4B,E) in both wild-type and Ca V 1.3 −/− mice, respectively. In WT type II hair cells the amplitude of the Cd 2+ -sensitive current (I Ca : Figures 4A,C) was not significantly different to that obtained by leakage-subtraction (Figures 4B,C: P < 0.9336, F = 0.3916, DFn = 9, two-way ANOVA), or to the Cd 2+ -sensitive current measured in wild-type type I hair cells (Figures 3B,D: P < 0.8457, two-way ANOVA). The size of the Cd 2+ -sensitive I Ca recorded in type II hair cells of Ca V 1.3 −/− mice (Figures 4D,F) was very similar to that measured in type I hair cells (Figures 3F,H, P < 0.7677, F = 0.6319, DFn = 9, two-way ANOVA). In type II hair cells, the mean peak I Ca after Cd 2+ -subtraction was 142 pA in wild-type and 27 pA in Ca V 1.3 −/− mice (19.0% of the wild-type value).
We tested whether the size of I Ca changed with age in hair cells from wild-type and Ca V 1.3 −/− mice. The peak I Ca amplitude, FIGURE 5 | Calcium current in type I and type II hair cells as a function of postnatal age in mice. Peak of the Ca 2+ current measured as a function of postnatal age in type I and type II hair cells from both wild-type and Ca V 1.3 −/− mice. Single data points are shown.
which was detected between −21 and −10 mV, was plotted as a function of postnatal day (Figure 5). The size of I Ca in both type I and type II hair cells appears to be comparable between early and late postnatal ages in both wild-type and Ca V 1.3 −/− mice.
Calcium Currents in the Inner Hair Cells of the Mouse Cochlea From Ca V 1.3 −/− Mice We investigated I Ca in cochlear IHCs by performing wholecells recordings at 32-36 • C and with the same solutions used for the above vestibular hair cell recordings (intracellular Cs + , extracellular TEA and 4AP, and 5 mM Ca 2+ (Figure 6). About 10% of the total I Ca in apical IHCs has been shown to be carried by Ca 2+ channels other than Ca V 1.3 (Platzer et al., 2000;Michna et al., 2003). Indeed, we found that the size of I Ca , measured after leakage-subtraction, was largely reduced in IHCs from Ca V 1.3 −/− mice compared to littermate controls at both immature (P < 0.0001, F = 63.70, DFn = 9) and adult ages ( Figure 6G, P < 0.0001, F = 20.33, DFn = 9, between the same voltage range used for the vestibular hair cells: −61 to +29 mV, two-way ANOVA). The nature of the residual Ca 2 channels in immature (∼16% of the wild-type value) and adult (∼12%) IHCs is unknown, but previous suggestions included Ca V 1.4 (Brandt et al., 2003) or, in immature avian auditory hair cells, Ca V 3.1 T-type subunits (Levic and Dulon, 2012).

DISCUSSION
In the present study, we compared the level of expression of the Ca 2+ current in hair cells of the crista and cochlear IHCs from wild-type and Ca V 1.3 −/− mice. We also investigated whether the normal developmental acquisition of the maturelike K + current in the crista hair cells was dependent on the presence of Ca V 1.3 Ca 2+ channels, as previous demonstrated for the IHCs (Brandt et al., 2003;Jeng et al., 2020a). We found that the adult-like K + currents in type I and type II hair cells were similarly present in both wild-type and Ca V 1.3 −/− mice. We also showed that the IHCs and crista hair cells from Ca V 1.3 −/− mice exhibit a residual Ca 2+ current that was only < 20% to that of control cells (ranging between 12 and 19% depending on hair-cell type and age investigated). The data provided have highlighted some differences in the way inner ear organs regulate the development of hair cells.
It is well established that the development of IHCs and outer hair cells (OHCs) in the cochlea is tightly linked to their ability to elicit Ca 2+ dependent action potentials during pre-hearing stages (Corns et al., 2014;Ceriani et al., 2019), where hearing onset occurs at P12 in most altricial rodents. One of the basolateral biophysical characteristics of cochlear hair cells, which is controlled by this spiking activity, is the expression of adult-like K + channels. Cochlear hair cells from pre-hearing Ca V 1.3 −/− mice, which are unable to elicit Ca 2+ action potentials retain an immature K + current profile even at adult stages (Brandt et al., 2003;Ceriani et al., 2019;Jeng et al., 2020a). We found that the absence of the Ca V 1.3 Ca 2+ current does not, however, influence the maturation of vestibular hair cells, most likely because these cells seem not to elicit action potentials during early postnatal developmental stages.
The absence of Ca V 1.3 Ca 2+ channels in both mice (Platzer et al., 2000;Dou et al., 2004) and humans (Baig et al., 2011) is associated with deafness but not visible vestibular dysfunctions. Thus, it is not clear why vestibular function is not suppressed or largely reduced by the absence of Ca 2+dependent exocytosis in hair cells. Different from hearing, partial vestibular compensation may occur by other sensory systems (e.g., proprioception and vision), so that specific vestibular tests such as vestibular evoked potentials (VsEPs) are likely to be required to unveil subtle vestibular impairments (Jones and Jones, 2014). This has been previously demonstrated for another synaptic protein, otoferlin, which is expressed in both cochlear and vestibular hair cells. Otoferlin knockout mice are deaf but do not show gross vestibular dysfunction (abnormal posturing, imbalance or nystagmus; Roux et al., 2006), although subtle vestibular deficits can be unveiled by VsEPs (Dulon et al., 2009). Similar to cochlear IHCs, type I and type II hair cells from the crista of Ca V 1.3 −/− mice exhibit a small (<20%) residual Ca 2+ current, indicating that the Ca V 1.3 subunit carries the large majority of the current. This result differs from a previous study performed on early postnatal hair cells from the utricle of Ca V 1.3 −/− mice (P1-P10: Dou et al., 2004), which exhibit a much larger (about 50%) residual inward Ba 2+ current through voltage-gated Ca 2+ channels. It is unlikely that age difference among studies is the reason for this discrepancy because we observed a similar residual Ca 2+ current in both neonatal and adult vestibular hair cells from Ca V 1.3 −/− mice. Tukey's post-test analysis: wild-type immature (−473 ± 112 pA, n = 9) vs. Ca V 1.3 −/− immature (−74 ± 27 pA, n = 17), P < 0.0001; wild-type adult (−243 ± 61 pA, n = 12) vs. Ca V 1.3 −/− adult (−28 ± 14 pA, n = 6), P < 0.001; wild-type immature vs. wild-type adult, P < 0.001; wild-type immature vs. Ca V 1.3 −/− adult, P < 0.001; Ca V 1.3 −/− immature vs. wild-type adult, P < 0.001; Ca V 1.3 −/− immature vs. Ca V 1.3 −/− adult, P > 0.05. Data are reported as mean ± SD.
An alternative explanation is that the expression level of the Ca V 1.3 subunit is different between utricle and crista hair cells. In principle, the residual Ca 2+ current in some type I and type II vestibular hair cells could at least in part reduce the vestibular phenotype in Ca V 1.3 −/− mice, provided that the remaining Ca 2+ channels are capable of driving some exocytosis. An attractive possibility for the absence of vestibular phenotypes in Ca V 1.3 −/− mice, however, is likely to be associated with the presence of non-quantal synaptic transmission in vestibular type I hair cells, but not cochlear IHCs (Yamashita and Ohmori, 1990;Holt et al., 2007;Songer and Eatock, 2013). This non-conventional, K + -based synaptic transmission relays on intercellular K + increase in the extended narrow synaptic cleft between the basolateral membrane of type I hair cells and the inner face of the afferent calyx terminal (Lim et al., 2011;Contini et al., 2012Contini et al., , 2017Spaiardi et al., 2020a) and fast, direct resistive coupling between pre-and postsynaptic K + channels facing the synaptic cleft (Contini et al., 2020). I K,L , which is normally present in Ca V 1.3 −/− mice, plays a major role in this K + -based signal transmission at the calyx synapse (Eatock, 2018). Indeed, we found that intercellular K + increase still occurs in Ca V 1.3 −/− mice (Figure 1), suggesting that calyces develop normally around the type I hair cell basolateral membrane. Non-synaptic activation of the calyx has also been described during the mechanical deflection of the hair bundle of the type I hair cells (Songer and Eatock, 2013). The presence of this K + -based afferent transmission, which is unlikely to be affected in Ca V 1.3 −/− mice, might contribute to the absence of obvious vestibular dysfunction in these mice.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.

ETHICS STATEMENT
The animal study was reviewed and approved by University of Sheffield Ethical Review Committee.

AUTHOR CONTRIBUTIONS
WM and SM conceived and coordinated the study. All authors helped with the collection and analysis of the data.

FUNDING
This work was supported by the BBSRC (BB/T004991/1) to WM and University of Pavia to SM.