(A) The Clrn1-floxed mice (Clrn1^ex4fl/fl) were engineered by addition of loxP sites on either side of exon 4. Clrn1^ex4fl/fl mice were crossed with PGK-Cre^+/– mice to achieve the early and ubiquitous elimination of clarin-1 (Clrn1^ex4–/– mice), and with Myo15-Cre^+/– mice to target clarin-1 elimination specifically to hair cells at postnatal stages (Clrn1^ex4fl/fl Myo15-Cre^+/– mice). A topological representation of the murine clarin-1, a protein containing 4 transmembrane domains with a C-terminal type II PDZ-binding motif (PBM, boxed), is shown. (B) RT-PCR shows mRNA expression of Clrn1 isoforms 1, 2, and 3 in the organ of Corti (P30) and only isoform 2 and 3 in P30 WT IHCs. Note that the expression of all isoforms is absent in P30 IHCs from Clrn1^ex4fl/fl Myo15-Cre^+/– (see uncut gels in supplemental material). (C) ABR thresholds in Clrn1^ex4fl/fl Myo15-Cre^+/– mice (light orange to dark red curves; mean ± SD) show a progressive increase between P15 and P18. A gray curve (control mice) and a blue curve (Clrn1^ex4–/– mice) for P15–P20 are also shown. (D) In Clrn1^ex4fl/fl Myo15-Cre^+/– mice (red), ABR thresholds (mean ± SD) progressively increase to reach severe deafness at P21–P28 as compared with those in control mice (gray-black). (E) DPOAEs are absent in Clrn1^ex4–/– mice (blue) as early as P15. By contrast, Clrn1^ex4fl/fl Myo15-Cre^+/– mice (red) display unaffected DPOAE thresholds up to P20 (green area). From P20 onward, DPOAE thresholds slowly increase by about 20–30 dB SPL. (F) Compound action potential (CAP) responses on P15–P19. CAPs were produced by 10-kHz, 95-dB-SPL, tone bursts. Bar charts represent CAP wave I latency and amplitude, which are significantly delayed and reduced, respectively, in Clrn1^ex4fl/fl Myo15-Cre^+/– mice. *P < 0.05 (2-tailed unpaired t test with Welch’s correction, n in the figure).

(A–F) Hair bundle structure is affected in Clrn1^ex4–/– mice but not in Clrn1^ex4fl/fl Myo15-Cre^+/– mice. Throughout the spiral cochlea, the OHC hair bundles of Clrn1^ex4–/– mice are linear, wavy, and/or hooked in shape (B and E), contrasting with the V-shaped hair bundles in control (A and D) and Clrn1^ex4fl/fl Myo15-Cre^+/– (C and F) mice. Note that the upper image of IHCs in C is a crop of (lower portion of P20 panel). Similar hair bundle abnormalities were observed along the entire cochlear axis between P12 and P25 (images representative of 12 mice). The stereocilia of the short row (colored in red) regressed entirely in most of the OHCs of P12 Clrn1^ex4–/– mice (E), but not in those of P20 Clrn1^ex4fl/fl Myo15-Cre^+/– mice (F). (G) The physical coupling between the tallest stereocilia of OHCs and the overlying tectorial membrane (TM) was preserved in both Clrn1^ex4–/– and Clrn1^ex4fl/fl Myo15-Cre^+/– mice, as shown by the presence of imprints of the stereocilia (white arrows) on the lower surface of the tectorial membrane. Scale bars: 1 μm.

(A) Distribution of USH1 proteins (myosin VIIa, harmonin b, cadherin-23, protocadherin-15) and stereocilin in the apical region of F-actin–labeled stereocilia (red) in P12 control mice. (B) The typical targeting and localization of USH1 proteins in the stereocilia is preserved in the absence of clarin-1, regardless of the shape of the Clrn1^ex4–/– hair bundles. (C) The F-actin labeling of the cuticular plate in P12 Clrn1^ex4–/– mice is irregular, with the presence of furrows delimiting regions of clumped stereocilia. Stereocilin immunostaining (green) was used to visualize the hair bundle stereocilia. (D) No such irregularity of staining is observed in OHCs from control and Clrn1^ex4fl/fl Myo15-Cre^+/– mice. Scanning electron microscopy micrographs show selected OHC hair bundles (from P12 Clrn1^ex4–/–, P15 control, and P20 Clrn1^ex4fl/fl Myo15-Cre^+/– mice) with hair bundle shapes similar to those of the immunostained hair bundles. Images obtained from 2–5 mice for each genotype. Scale bars: 1 μm.

(A–D) Ca²⁺ currents in IHCs were activated with a depolarizing voltage-ramp protocol (1 mV/ms) from –90 to +30 mV. The amplitude of Ca²⁺ currents is normal on P9, but larger in Clrn1^ex4–/– (blue curves, A and B) and Clrn1^ex4fl/fl Myo15-Cre^+/– (red curves, C and D) mice than in controls (black curves, A–D) on P13. (E) Comparative change in peak Ca²⁺ current density (peak I_Ca2+ at –10 mV normalized with respect to cell size) with age, at pre- and post-hearing stages (from P6 to P18), in IHCs from Clrn1^ex4fl/fl Myo15-Cre^+/– mice and control mice. (F) Boltzmann fit of the I-V curve for IHC Ca²⁺ currents (100-ms voltage steps) in Clrn1^ex4fl/fl Myo15-Cre^+/– and control mice on P18. (G) Comparative change in the half-maximal activation voltage of I_Ca2+ (V_1/2) before and after hearing onset (from P6 to P18) in IHCs from Clrn1^ex4fl/fl Myo15-Cre^+/– and control mice. (H and I) I_Ca2+ activation time constant measured for various voltage steps from a holding potential at –80 mV in IHCs of P13 Clrn1^ex4–/– mice (H), P18 Clrn1^ex4fl/fl Myo15-Cre^+/– mice (I), and the corresponding control mice. (J) IHCs from P18 Clrn1^ex4fl/fl Myo15-Cre^+/– (red) and control (black) mice were subjected to voltage steps from –80 mV to various membrane potentials for 100 milliseconds (inset: current traces for a Clrn1^ex4fl/fl Myo15-Cre^+/– mouse and a control mouse). The current reduction at the end of the 100-millisecond step (Ca²⁺-dependent inactivation, CDI) is expressed as a percentage of I_Ca2+ peak (%): CDI = [I_{Ca2+ peak} – I_{Ca2+ 100 ms}]/I_{Ca2+p eak}. The values shown are means ± SEM. *P < 0.05 (E–J, Student’s t test with Welch’s correction; and A–D, 2-way ANOVA); n, number of cells.

(A–D) Exocytosis was measured after various voltage steps between –80 mV and –5 mV, with each voltage step lasting 100 milliseconds (A–C) or 25 milliseconds (D). A simple power function was fitted to the data (y = ax^N, where a is exocytotic slope efficiency and N the power index; ). Ca²⁺ efficiency for IHC exocytosis was normal at P9, but was significantly lower in P13 Clrn1^ex4–/– mice (a = 0.04 ± 0.01 fF/pA) than in control littermates (a = 0.13 ± 0.01 fF/pA; P < 0.05) and in P13 Clrn1^ex4fl/fl Myo15-Cre^+/– mice (a = 0.07 ± 0.01 fF/pA) than in control littermates (a = 0.16 ± 0.01 fF/pA; P < 0.05). (E–G) Mean exocytotic response curves evoked by UV uncaging of intracellular Ca²⁺ in IHCs from P14 Clrn1^ex4fl/fl Myo15-Cre^+/– (red) and control (black) mice. (F) Exocytosis rates obtained by a derivative function (dC_m/dt) of the curves shown in E. (G) Comparative exocytotic peak upon Ca²⁺ photorelease in mutant and control IHCs. (H) Exocytosis kinetics for constant voltage steps from –80 to –10 mV, with durations increasing from 10 to 100 milliseconds, in IHCs from P18 Clrn1^ex4fl/fl Myo15-Cre^+/– (red) and control (black) mice. The intracellular Ca²⁺ buffer was 1 mM or 5 mM EGTA. A simple exponential function was fitted to the data, with similar time constants of 25 ± 10 milliseconds (n = 12) and 33 ± 18 milliseconds (n = 11) for controls in 1 mM and 5 mM EGTA, respectively. For Clrn1^ex4fl/fl Myo15-Cre^+/– IHCs, the time constant increased from 102 ± 50 milliseconds (n = 27) in 1 mM EGTA to 205 ± 32 milliseconds (n = 10) in 5 mM EGTA (P < 0.05). The values shown are means ± SEM; *P < 0.05 (A–F and H, 2-way ANOVA; and G, Mann-Whitney test).

(A) Two representative IHCs of P20 control, Clrn1^ex4fl/fl Myo15-Cre^+/–, and Clrn1^ex4–/– mice stained for otoferlin (red) and ribeye (green), showing the correct location of the ribbons in the basolateral region of mutant IHCs despite the absence of clarin-1. (B) The bar chart shows the number (mean ± SEM; unpaired Student’s t test) of synaptic ribbons per IHC, which is similar in P20 Clrn1^ex4–/– (blue), Clrn1^ex4fl/fl Myo15-Cre^+/– (red), and control (black) mice. (C) In control IHCs, efferent terminals (artificially colored in light blue) contact only afferent terminals (green). (D) By contrast, in Clrn1^ex4–/– mice, efferent nerve fibers (blue) are found in direct contact with IHCs, on both P15 and P28. (E) Round, oval, and droplet-shaped ribbons are present in mutant IHCs lacking clarin-1 on P15. Each transmission electron microscopic image in C–E is representative of 5–10 IHCs analyzed from 3 different mice for each genotype. (F) The bar chart shows the percentage of immature and mature synaptic ribbons per IHC in Clrn1^ex4–/– (blue platform), Clrn1^ex4fl/fl Myo15-Cre^+/– (pink platform), and control (gray platform) mice. The IHCs of Clrn1^ex4fl/fl Myo15-Cre^+/– mice have normal frequencies of round (immature) and droplet-shaped (mature) ribbons on P15 (E and F). Similar values were obtained on P15 and P28. In the IHCs of Clrn1^ex4–/– mice, round ribbons were numerous on P15, but their frequency had decreased to normal values on P28 (F). **P < 0.01 (Mann-Whitney test). Scale bars: 5 μm (A), 100 nm (C, D, and E).

(A) The number of Ca_v1.3-immunoreactive patches associated with ribbons per IHC is similar in control (black) and Clrn1^ex4–/– (blue) mice, on both P9 and P13. The numbers of Ca_v1.3 patches counted are indicated in parentheses. (B) Representative Imaris 3D images and quantitative analysis (bar chart) of immunoreactive patches in the synaptic basal region of IHCs from P13 control (black) and Clrn1^ex4–/– (blue) mice. Ca_v1.3 channels form larger patches in the active zone, and the ribbons are smaller in IHCs lacking clarin-1 than in control IHCs. (C) The rate of colocalization for Ca_v1.3- and ribeye-immunoreactive areas is normal on P9, but low on P13 in the absence of clarin-1. (D) Representative confocal images of Ca_v1.3-immunoreactive (green) and ribeye-immunoreactive (red) patches, and a line scan analysis (intensity profile for a single Ca_v1.3- and ribeye-associated patch) in P15 control, P15 Clrn1^ex4–/–, and P18 Clrn1^ex4fl/fl Myo15-Cre^+/– mice (images representative of 6 mice for each genotype). (E and F) Cytoskeleton architecture in IHCs from control and Clrn1^ex4–/– mice. The F-actin cortical network (purple) is altered in the IHC synapse region of Clrn1^ex4–/– on P9 (E) and P13 (F). The bar charts show the mean values ± SEM; *P < 0.05 (unpaired Student’s t test). Scale bars: 1 μm.

(A) The schematic diagram (top panel) shows the AAV2/8-Clrn1-IRES-GFP vector used for gene delivery to the cochlea on P2–P3. The histogram indicates the percentage of GFP-labeled IHCs and OHCs on P20–P30 in Clrn1^ex4–/– mice. (B) Injections of either AAV2/8-Clrn1-IRES-GFP or AAV2/8-GFP had no effect on click ABR thresholds in WT mice. Injections of AAV2/8-GFP also had no impact on ABR thresholds in Clrn1^ex4fl/fl Myo15-Cre^+/– mice (unpaired Student’s t test). (C) ABR thresholds (mean ± SEM) in treated (purple, n = 9) and untreated (blue, n = 9) inner ears of P22–P24 Clrn1^ex4–/– and control (black, n = 5) mice. A decrease of about 10–15 dB in ABR thresholds relative to untreated ears was observed in Clrn1^ex4–/– ears after injection. **P < 0.01 (unpaired Student’s t test). (D) Early postnatal AAV2/8-mediated delivery of clarin-1 into Clrn1^ex4–/– ears did not prevent or correct the misshaping of the hair bundles. Both IHC and OHC hair bundles presented alterations in shape and a loss of short-row stereocilia due to the embryonic loss of clarin-1 (representative of 8 mice between P25 and P30). Scale bars: 1 μm.

(A) Comparative tone-burst ABR thresholds (audiogram, mean ± SEM and individual points for each frequency) in AAV2/8-Clrn1–injected (purple, n = 7) and untreated (red, n = 10) inner ears of P22–P24 Clrn1^ex4fl/fl Myo15-Cre^+/– mice and control (black, n = 6) mice. (B) Similar comparative audiograms for AAV2/8-Clrn1–injected (purple, n = 5) and untreated (red, n = 6) inner ears of P60 Clrn1^ex4fl/fl Myo15-Cre^+/– mice and P60 control (black, n = 6) mice. (C) Mean click ABR waves at P60 in control and in untreated and AAV2/8-Clrn1–injected Clrn1^ex4fl/fl Myo15-Cre^+/– mice. (D) Scanning electron microscopy view of stereocilia from untreated (left) and AAV2/8-Clrn1–injected (right) ears of P120 Clrn1^ex4fl/fl Myo15-Cre^+/– mice (n = 3). By contrast to the untreated ears, the injection of AAV2/8-Clrn1 resulted in apparently normal hair bundles still visible on P120 (n = 3). However, despite the persistence of V-shaped bundles, the stereocilia in the short and middle rows are heterogeneous in length, and many of these stereocilia are missing. **P < 0.01, ***P < 0.001 (unpaired Student’s t test).Scale bars: 1 μm.

(A) The re-expression of clarin-1 at the IHC synapse (P15–P18) in Clrn1^ex4–/– IHCs led to normal Ca²⁺ currents, kinetics, and Ca²⁺ efficiency of exocytosis (). *P < 0.05 (2-way ANOVA). (B) The virus-mediated expression of clarin-1 restored the tight clustering of Ca²⁺ channels in the IHC active zone, as shown by the line scan intensity profiles of Ca_v1.3- and ribeye-immunoreactive patches for 2 ribbons (images representative of 3 mice).

(A and B) The GST-tagged clarin-1 N-terminal region (Clrn1-N) and C-terminal region (Clrn1-C) bind to CFP-tagged Ca_Vβ₂ produced in HEK293 cells, whereas GST alone does not. No binding is detected with the untagged Ca_Vα₁, CFP-tagged Rab11, or ECFP alone. (B) The bar chart indicates the significant interaction between clarin-1 and Ca_Vβ₂ (n = 5, *P < 0.05; unpaired Student’s t test). (C and D) In a coimmunoprecipitation assay, anti-FLAG M2 resin was incubated with HEK293 cells coproducing FLAG-tagged Ca_VAID, mCherry-tagged clarin-1, and CFP-tagged Ca_Vβ₂, or mCherry-tagged clarin-1 and CFP-tagged Ca_Vβ₂. (D) Significant binding between Ca_VAID and clarin-1 is observed only in the presence of Ca_Vβ₂ (n = 3, *P < 0.05; unpaired Student’s t test). (E) GST-tagged Clrn1-C binds to myc-tagged harmonin b, whereas GST alone does not. The bar chart shows the significant interaction between clarin-1 and harmonin (n = 5, P < 0.05; unpaired Student’s t test). (F) Schematic representation of a synaptic active zone summarizing the interactions between clarin-1, the Ca²⁺ channel subunits Ca_Vβ₂ and Ca_Vα₁, and harmonin (see uncut gels in supplemental material).

(A) Representative electrically evoked brainstem responses (EEBRs) in the cochleae of P20 control, Clrn1^ex4fl/fl Myo15-Cre^+/–, and Clrn1^ex4–/– mice. The first large downward inflection is due to an electrical artifact. In these stimulation conditions, wave I is not visible, as signal processing by the IHC synapse is bypassed. EEBR wave II (EII) and later waves (EIII and EIV), corresponding to the responses of higher auditory centers, are clearly visible in the control mouse (black trace, representative of 10 mice), absent in Clrn1^ex4–/– mice (blue trace, representative of 6 mice), and significantly delayed and reduced in Clrn1^ex4fl/fl Myo15-Cre^+/– mice (red trace, representative of 10 mice). (B) Cochleae immunostained with Neurofilament 200 (NF200; green) showing a significant loss of auditory nerve fibers in P25 Clrn1^ex4–/– mice (arrows) relative to control and Clrn1^ex4fl/fl Myo15-Cre^+/– mice of the same age. (C) IHCs of P20 control and Clrn1^ex4fl/fl Myo15-Cre^+/– mice. The postsynaptic GluA2/3-immunoreactive domain (green) is abnormally expanded in the nerve terminals underneath the IHCs of Clrn1^ex4fl/fl Myo15-Cre^+/– mice (n = 7). (D) Representative micrographs of the IHC synaptic region highlighting the expansion of postsynaptic terminals in Clrn1^ex4fl/fl Myo15-Cre^+/– mice (red dotted line; representative of 3 mice and 10 IHCs) and the swelling (artificially colored in green) of the postsynaptic boutons in Clrn1^ex4–/– mice (representative of 3 mice and 10 IHCs) as compared with control mice (representative of 3 mice and 13 IHCs). (E) Quantification of the loss of parvalbumin-positive neurons in Rosenthal’s canal in control (4 cochleae from 3 mice), Clrn1^ex4–/– (8 cochleae from 4 mice), and Clrn1^ex4fl/fl Myo15-Cre^+/– mice (6 cochleae from 3 mice) (values are mean ± SD; ANOVA, post hoc Holm-Sidak test). Scale bars: 500 nm (D), 20 μm (B and E), 5 μm (C).

(A) Schematic diagram of an IHC synaptic active zone in control (top panel) and clarin-1–deficient (bottom panel) mice. In the presynaptic active zone of release, clarin-1 interacts with Ca_Vβ₂, harmonin, and the F-actin cytoskeleton to cluster Ca_v1.3 Ca²⁺ channels close to the synaptic vesicles. The lack of clarin-1 results in a dislocation of the synaptic F-actin network associated with a spatial disorganization of both presynaptic Ca_v1.3 channels and postsynaptic GluA2/3 glutamate receptors. (B) Schematic representation of molecular interactions between clarin-1 and other proteins in the IHC synaptic active zone. Clarin-1 may be required for the trans-synaptic alignment of the Ca_v1.3 channel complex (harmonin-Ca_vβ₂-Ca_Vα₁) and postsynaptic glutamate receptors.