Animal handling complied with national animal care guidelines. For rats and chicken, handling was approved by the Administrative Panel on Laboratory Animal Care (APLAC) of Stanford University and accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care (#A3213-01). For mice, handling was approved by the University of Göttingen Board for animal welfare and the animal welfare office of the state of Lower Saxony, Germany.

Utricles were dissected from embryonic day 18 (E18) old chicken. Otoconial membranes and spiral ganglion nerve fibers were removed and utricles were collected into chilled lysis buffer (50 mM Tris-HCl, 140 mM NaCl, 5% Glycerol, 250 mM sucrose, protease inhibitors (Roche, EDTA-free), pH 7.4). The tissue was homogenized by triturating six times through a 26 gauge needle, followed by centrifugation for 5 min at 600 × g at 4°C (Eppendorf tabletop Centrifuge 5417C). The supernatant was subsequently centrifuged at 100000 × g for 30 min at 4°C to pellet membranes (Beckmann, TL-100 Ultracentrifuge). The pellet was resuspended in solubilization buffer (20 mM Tris-HCl, 10% glycerol, 140 mM NaCl, 1% octyl-β-D-glucopyranoside, protease inhibitors, pH 7.4) for 1 h on ice. After solubilization, samples were centrifuged for 20 min at 55000 × g at 4°C (Beckmann, TL-100 Ultracentrifuge). Supernatants were incubated with 25 μL Dynabeads (Life Technologies, Dynabeads M-270 Epoxy) conditioned with either 5 μg chicken HCS-1 antibody (mouse, monoclonal; Goodyear et al., 2010) or 5 μg control antibody, TLA (tip-link antigen, mouse monoclonal; Goodyear and Richardson, 2003), according to the manufacturer’s protocol. Immunoprecipitation of otoferlin was performed for 2 h at 4°C. Beads were washed three times for 15 min at 4°C with solubilization buffer containing 0.1% octyl-β-D-glucopyranoside. Beads were then boiled for 5 min at 95°C in Laemmli sample buffer (BioRad), loaded onto a 4–20% SDS PAGE (BioRad) and proteins were allowed to run 1 cm into the separation gel and visualized with Coomassie brilliant blue staining (BioRad).

Gel bands were excised and digested in-gel using trypsin (Promega) as previously described (Shevchenko et al., 2006). Dried peptides were reconstituted in 0.1% formic acid, 2% acetonitrile and 97.9% water. Peptides were loaded onto a self-packed C18 reverse phase column with an ID of 100 μM and 15 cm in length. Over the course of all LC-MS/MS experiments, two LCs were used: a nanoAcquity UPLC (Waters) and a nano2D LC (Eksigent, AB Sciex), with flow rates of 300 nL/min and mobile phase A consisting of 0.585% (vol/vol) acetic acid in water and mobile phase B of 0.585% (vol/vol) acetic acid and 2% (vol/vol) water in acetonitrile. The mass spectrometer (LTQ Orbitrap Velos, Thermo Fisher) utilized data-dependent acquisition in which the top 12 most intense precursor ions were selected for fragmentation. The raw data were converted to mzXML format and searched against the UniProt Gallus gallus database using Sequest on a sorcerer platform (Sage-N). Search parameters included tryptic specificity, allowing for a maximum of two miscleavages with static modification of propionamide (cysteine) and variable modifications of oxidation (methionine), phosphorylation (serine, threonine, tyrosine) and alkylation (lysine). The precursor mass tolerance was 20 ppm, and the data was further filtered using the Scaffold software (Proteome Software). In the case of identified interacting partners we stringently required at least 4 unique peptides with a 95% peptide threshold and 99.9% protein probability threshold, thereby effectively filtering out all non-specific contaminants.

PCRs on a few IHCs were performed essentially as described (Kerr et al., 2008; Reisinger et al., 2011). Organs of Corti (OCs) from P14 mice were dissected in HEPES-Hanks solution (5.36 mM KCl, 141.7 mM NaCl, 1 mM MgCl₂, 0.5 mM MgSO₄, 10 mM Na-HEPES, 6.84 mM L-glutamine, 5.55 mM D-glucose, pH 7.2) and perfused with modified Ringer’s solution thereafter (113 mM NaCl, 35 mM TEA-Cl, 2.8 mM KCl, 2 mM CaCl₂, 1 mM MgCl2, 10 mM Na-HEPES, 1 mM CsCl, 11.1 mM D-glucose; pH adjusted to 7.2, osmolarity approximately 300 mOsm). Outer hair cells and supporting cells were removed with glass capillaries in a patch-clamp setup. Once inner phalangeal cells were removed, 3–5 IHCs were collected in one patch clamp glass capillary filled with 8 μL KCl solution (140 mM KCl, 5 mM K-HEPES, 5 mM EGTA, 3 mM MgCl₂, pH 7.3). Before and after collecting cells, bath controls were taken by lowering the patch pipette close to the tissue and removing overpressure for 5–10 s. The content of each capillary was expelled into a reaction tube containing buffer for reverse transcription [2.5 μL first strand buffer, 0.6 μL Oligo(dT)₂₀ primer (50 μM), 0.5 μL Random hexamers (50 ng/μL), 0.7 μL dNTP mix (10 mM each), 1.4 μL DTT (0.1 M) and 0.8 μL Ribonuclease Inhibitor (40 U/μL)]. The reaction was started by adding 0.5 μL SuperScript^® IV Reverse Transcriptase (100 units; Thermo Fisher Scientific) and incubated for 10 min at room temperature (RT), followed by 20 min at 37°C and 2 h at 42°C. The resulting cDNA was precipitated over night at -20°C in 70% EtOH and 1 μL glycogen (Ambion), washed with 70% EtOH, dried and resuspended in 25 μL H₂O. The cDNA solution from each sample was split into six PCR reactions. cDNA quality was assessed with TaqMan assays for bassoon (Mm00464451_m1; Applied Biosystems) and TATA-binding protein (Mm00446973_m1; Applied Biosystems). To test for CaMKII isoforms in a SYBR green assay, we designed intron-overspanning amplicons targeting all splice variants of the four CaMKII genes (in Mus musculus) described as reference sequences in NCBI databases. Amplification efficiency of the assays was determined with standard curve assays, resulting in 95.7–96.9% efficiencies for all transcripts. We used the following oligonucleotides: CaMKIIα: 5′-GAAGATGTGCGACCCTGGAA-3′ and 5′-TGATGCGGATATAGGCGATG-3′ (400 nM each), CaMKIIβ: 5′-ACAAACAGCACCAAAAACAGCT-3′ and 5′-GAGCTGCTCTGTGGTCTTGA-3′ (300 nM each), CaMKIIγ: 5′-TTACGCAAATTCAACGCCCG-3′ and 5′-GACACCGCCATCTGACTTCT-3′ (400 nM each), CaMKIIδ: 5′-CGTCTCTTGAAGCACCCCAA-3′ and 5′-AAACAGTTCGCCACCAGTCA-3′ (300 nM each). Mouse brain cDNA was used as positive control. Amplification with 2x TaqMan universal PCR Mastermix (Applied Biosystems) or 2x Power SYBR green Mastermix (Applied Biosystems) was conducted in an Applied Biosystems 7500 Real Time PCR system using default PCR parameters. Dissociation curve assays revealed the melting temperatures to control for amplicon specificity. In addition, we assayed the size of all amplicons with gel electrophoresis on 2% agarose gels with EtBr staining. We analyzed only those IHC samples for CaMKII expression where both bassoon and TATA-binding protein transcripts were detected.

Sprague Dawley rats at P9 to P11 or P14 C57Bl6J mice of either gender were decapitated and cochleae were dissected in chilled Hank’s balanced salt solution (HBSS, HyClone). OCs were fixed in chilled 3% paraformaldehyde for 25 min, permeabilized with 0.5% Triton X-100 in PBS for 30 min and incubated in blocking solution (1% BSA and 0.1% Triton X-100 in PBS) for 1 h at RT. Antibodies to otoferlin (mouse, 1:400; Abcam, ab53233), myosin VI (rabbit, 1:400; Sigma, M5187), parvalbumin (1:2000, raised against bullfrog parvalbumin-3, in mice it recognizes oncomodulin and parvalbumin; Heller et al., 2002), CaMKIIα (rabbit, 1:300; Sigma–Aldrich, C6974), CaMKIIβ (rabbit, 1:300; Abcam, ab34703), CaMKIIγ (rabbit, 1:300; Acris, AP13886PU-N), CaMKIIδ (rabbit, 1:300; Genetex, GTX111401), pan-CaMKII (rabbit, 1:300; Abcam, ab52476), PSD95 (mouse, 1:500, Abcam, ab2723), Ctbp2 (goat, 1:200, Santa Cruz, sc-5966) and phosphoserine (rabbit, 1:300; Abcam, 9332), were diluted in blocking solution and incubated on OCs overnight at 4°C. Alexa fluor 488, 546, 568, and 633-conjugated secondary antibodies (1:200-1:600; Thermo Fisher Scientific) were used. The tissue was mounted with Aqua Poly/Mount (Polysciences) and for Figures 3, 4, and 10 imaged using an AxioImager/LSM 5 Exciter confocal microscope (Zeiss). Images in Figure 1 were acquired using a Leica SP5 confocal microscope with 63x glycerol objective (NA 1.456).

P9–P11 rat OCs were dissected in chilled external solution (10 mM HEPES, 2 mM MgCl₂, 2 mM CaCl₂, 2 mM KCl, 145 mM NaCl, 6 mM D-glucose, 2 mM ascorbate, 2 mM pyruvate, 2 mM creatine, pH 7.4) and fixed in chilled 3% paraformaldehyde for 25 min. For hair cell stimulation, acutely dissected OCs were transferred into prewarmed high K⁺ external solution (KCl increased to 40 mM, NaCl reduced to 35 mM) and incubated for 15 min at 37°C and 5% CO₂ before fixation. To pharmacologically inhibit CaMKII, OCs were incubated for 10 min in prewarmed external solution supplemented with 50 μM of selective inhibitor KN-93 (Cayman Chemical) followed by stimulation with high K⁺ external solution + KN-93 and fixation. A proximity ligation assay (PLA) (Duolink, Sigma) was performed with mouse otoferlin (1:500; Abcam, ab53233) antibody in combination with rabbit myosin VI (1:500; Sigma, M5187), rabbit parvalbumin (1:2000; Heller et al., 2002), rabbit CaMKIIδ (1:300; Genetex, GTX111401), rabbit phosphoserine (1:300; Abcam, 9332), and rabbit pan-CaMKII (1:300; Abcam, ab52476). The manufacturer’s protocol was applied with the following modifications: Fixed OCs were semipermeabilized in 0.5% Triton X-100 at RT for 30 min and subsequently blocked with Blocking buffer (Duolink, Sigma) for 2 h at RT. Primary antibodies were diluted in antibody diluent (Duolink, Sigma) and incubated overnight at 4°C. The tissue was then washed four times with gentle shaking in 2 mL Buffer A (Duolink, Sigma). PLA probes (anti mouse MINUS and anti-rabbit PLUS; Duolink, Sigma) were diluted 1:6 in 30 μL antibody diluent and incubated for 1 h at 37°C and 5% CO₂. Tissue was washed four times with 2 mL Buffer A and gentle shaking, followed by incubation with 30 μL ligation mix for 30 min at 37°C and 5% CO₂. After three washes for 10 min in 2 mL Buffer A and gentle shaking, 30 μL DNA amplification mix was added and incubated for 100 min at 37°C and 5% CO₂. After amplification, the tissue was washed twice for 10 min in 1x Buffer B (Duolink, Sigma) followed by two times washing in 2 mL 0.01x Buffer B. To visualize hair cells, tissue was subsequently stained with Alexa Phalloidin 488 (Invitrogen) (1:200 in 0.01x Buffer B) for 15 min at RT, followed by two 10 min washing steps in 2 mL 0.01x Buffer B. For counterstainings of synaptic ribbons, anti-Ctbp2 antibody (goat, 1:200, Santa Cruz, sc-5966) was incubated together with primary antibodies to otoferlin and phosphoserine, and secondary anti-goat antibodies were co-incubated with PLA probes. For imaging, tissue was mounted with DAPI containing Duolink In Situ Mounting media. Images were taken with a LSM700 confocal microscope (Zeiss) with Zen software (Zeiss).

To quantify PLA signal intensities in confocal images, IHCs were outlined manually using Image J software and pixel intensities of the fluorescent PLA signals were determined. For each experimental condition three independent experiments were performed and a total of 30 IHCs were analyzed. The mean pixel intensity per IHC was calculated for each condition and compared. A two-tailed t-test was applied to assess a statistical significance of the changes in PLA signal pixel intensities amid the different experimental conditions.

Two mouse otoferlin fragments (C₂ABC: aa 1–632, 70 kDa and C₂DEF: aa 933–1920, 114 kDa) were PCR amplified from cDNA encoding full-length mouse otoferlin (NM_001100395) with a C-terminal HA-tag using the following primer pairs: C₂ABC-HA (5′-GAATTCACCATGGCCCTGATTGTTCACCT-3′, 5′-GCGGCCGCCTAAGCGTAATCTGGAACATCGTATGGGTACATGGTTCCTCCTGTGCAGCTCTCCGAGACAG-3′); C₂DEF (5′-GAATTCACCATGAGCAAGCAG-CGAAAGGACTTC-3′, 5′-GCGGCCGCCTAAGCGTAATCTGGAACATCGTATG-GGTACATGGTTCCTCCGCGAGCCAGGCCCACAGGG-3′). Fragments were subcloned into the pCl mammalian expression vector (Promega) via NotI and EcoRI. Full-length CaMKIIδ (NM_001025438.1) was amplified from mouse postnatal day 6 (P6) cochlea cDNA (5′-GAATTCACCATGGCTTCGACCACCACCTG-3′, 5′-GGTACCCCGATGTTTTG-CCACAAAGAGG-3′) and subcloned as N-terminal fusion construct into the pmCherry-N1 mammalian expression vector (Clontech) via EcoRI and KpnI. For co-immunoprecipitation (co-IP) of otoferlin, HEK293 cells were grown in a 10 cm culture dish to 80% confluency and transiently co-transfected with HA-tagged C₂ABC, HA-tagged C₂DEF and mcherry-tagged CaMKIIδ. 40 h post-transfection, cells were collected in TBS buffer (25 mM Tris-HCl, 150 mM NaCl, protease inhibitors (Roche, complete, EDTA free), pH 7.4), lysed by triturating 5 times through a 26^1/2 gauge needle, and centrifuged for 5 min at 4°C at 500 × g to remove debris. For co-IP of CaMKIIδ-mcherry with otoferlin C₂ABC-HA and/or C₂DEF-HA, the lysate was mixed with anti-HA agarose slurry (35 μg anti-HA antibody, Pierce HA Tag IP/Co-IP Kit, Thermo Scientific) and incubated with gentle end-over-end mixing for 2 h at 4°C. Agarose was washed four times with TBS-T buffer (25 mM Tris-HCl, 150 mM NaCl, 0.05% Tween, pH 7.4) before boiling for 5 min at 95°C in Laemmli sample buffer and applied on a 4–20% SDS PAGE gel (BioRad). Protein complexes were analyzed by immunoblots using a Trans Blot semi-dry transfer cell (BioRad) and polyclonal anti-HA antibodies (1:1000; Rockland, 600-401-384) and monoclonal anti-RFP antibodies (1:20000; Rockland, 200-301-379). Secondary anti-rabbit Dylight680 and anti-mouse Dylight800 antibodies (1:10000; Rockland, 611-144-003 and 610-145-003) were incubated for 1 h at RT, and after washing the blots for three times with TBS-T buffer, fluorescent signals were detected using a Li-Cor Odyssey system.

Two soluble mouse otoferlin domains comprising either the first three C₂ domains (C₂ABC), or the last three C₂ domains (C₂DEF) were heterologously expressed in Escherichia coli (E. coli) SoluBL21 (DE3). The C₂ABC fragment (aa 1–616, 70 kDa; NP_001093865) was PCR amplified (5′-AGCGGCTCTTCAATG-ATGGCCCTGATTGTTCACCT-3′, 5′-AGCGGCTCTTCTCCC-CTCCGAGACAGGCGTGGC-3′) and subcloned with a C-terminal hexahistidine-tag into the bacterial expression vector pPSG-IBA33 (Iba Lifesciences) and expressed at 30°C. After induction with IPTG, the temperature was changed to 16°C and the culture was harvested 16–20 h post-induction.

The cells were collected in lysis buffer (70 mM HEPES pH 7.4, 300 mM NaCl, 10 mM imidazole) and lysed by fluidizing (microfluidizer S, Microfluidics, Westwood, MA, United States). After centrifugation at 20000 rpm (JA-20 fixed angle rotor, Beckmann Coulter) for 45 min at 4°C, the supernatant was loaded onto a Ni-NTA-column (GE Healthcare). After washing, the recombinant proteins were eluted by gradient elution with imidazole containing buffer (70 mM HEPES, 300 mM NaCl, 500 mM imidazole, pH 7.4). For buffer exchange to 10 mM HEPES, 300 mM NaCl, pH 7.4, the proteins were further applied on a size exclusion chromatography column (HiPrep 16/60 Sephacryl S-200 HR, GE Healthcare). During all purification steps the temperature was kept at 4°C.

C₂DEF (aa 908–1932, 118 kDa; NP_001093865) was PCR amplified (5′-GAGAGGATCCAAGCTGGAGCTCTACCTGTG-3′, 5′-GAGAGAATTCTAATCA-GGTTCATTGCGAGCCAG-3′) and subcloned with a hexahistidine tag into the bacterial expression vector pET28a. Expression took place for 60 h at 16°C using an autoinduction system (Studier, 2005).

The C₂DEF was purified from inclusion bodies by resuspending the cell pellet in lysis buffer. After centrifugation for 45 min at 4°C and 20000 rpm (JA-20 fixed angle rotor, Beckmann Coulter), the supernatant was discarded and the pellet was washed three times with PBS buffer (4 mM KH₂PO₄, 16 mM Na₂HPO₄, 115 mM NaCl, pH 7.4), containing 1% Triton in the first washing step. The pellets were frozen overnight at -20°C and subsequently dissolved in Urea buffer (70 mM HEPES, 300 mM NaCl, 10 mM imidazole, 8 M urea, pH 7.4). After centrifugation, the supernatant was loaded onto a Ni-NTA-column (GE Healthcare) and recombinant proteins were eluted with an imidazole gradient.

For in vitro phosphorylation of recombinant otoferlin, 21.5 pmol recombinant CaMKIIδ (Life Technologies) was incubated together with equimolar amounts of recombinant otoferlin C₂ABC and C₂DEF domains (1:1:1) in 30 μL assay buffer (10 mM HEPES, 10 mM MgCl₂, 10 μg/mL calmodulin, 0.5 mM CaCl₂, 5 mM DTT, 100 μM ATP, pH 7.5) for 5 min at 30°C. The reaction was inactivated by adding Laemmli-buffer and subsequent incubation at 95°C for 5 min. For control experiments, otoferlin C₂ABC and C₂DEF were incubated in assay buffer in the absence of CaMKIIδ.

Gel bands corresponding to recombinant mouse otoferlin fragments, C₂ABC (70 kDa) and C₂DEF (118 kDa), were excised from the Coomassie gel after in vitro phosphorylation and prepared for LC-MS/MS analysis as described in above. During data acquisition the mass spectrometer was set to perform ion-trap MS/MS and high energy collision-induced dissociation (HCD) MS/MS on the same precursor masses to provide more complete fragmentation data and to increase the probability of correctly localizing the site of phosphorylation. All suggested phosphorylation sites were manually validated by interrogation of the fragment ion spectra, where neutral loss of phosphoric acid (H₃PO₄) was observed as well as site localization of the phosphor-group by corresponding b or y ions.

The protein fragments of otoferlin used for Ca²⁺ binding assays – C₂C (aa 410–616 in pGEX-6P-3, NP_001263649) and C₂F (aa 1695–1934 in pGEX-6P-3, NP_001263649.1) were expressed in E. coli Rosetta 2 (DE3) cells using the auto-induction system (Studier, 2005; Meese, 2015). The harvested cells were lysed in 75 mM HEPES pH 7.4, 300 mM NaCl using the microfluidizer S (Microfluidics, Westwood, MA, United States). The obtained supernatant after centrifugation was loaded onto 5 mL GST Trap columns (GE Healthcare). Using a glutathion containing buffer (75 mM HEPES pH 7.4, 300 mM NaCl, 25 mM reduced glutathion) the protein was eluted from the column and incubated with PreScission protease for 14 h at 4° C to cleave off the GST-tag. In the next step a size exclusion chromatography (S200 16/60, GE Healthcare) was performed (10 mM HEPES pH 7.4, 150 mM NaCl) followed by a GST trap column to separate the C₂ domain from the GST-tag. The protein solution was incubated with Chelex (Biorad) for 1 h at 4°C, concentrated and stored at -80°C.

In order to mimic phosphorylation sites, we replaced phosphorylated serine/threonine residues with aspartate residues. The cDNA for the “C2F-pm” fragment (S1783D, S1814D, T1866D) was newly synthesized by GeneArt (Life Technologies). For the “C2C-pm” fragment a mutation (T434D) was inserted using a “QuikChange” site directed mutagenesis protocol (Agilent Technologies). The expression and purification procedure was the same as for the wild type proteins.

For MicroScale Thermophoresis (MST) the NT.LabelFree instrument (NanoTemper Technologies GmbH, Munich, Germany) was used. All solutions were treated with Chelex (Biorad) to remove residual Ca²⁺. CaCl₂ (1M solution, Fluka) was diluted in size exclusion buffer (10 mM HEPES, 150 mM NaCl, 0.05% Tween, pH 7.4) and a series of 16 dilutions (1:2) was prepared and mixed with protein, resulting in ligand concentrations ranging from 0.6 μM to 200 mM. Proteins were used in concentrations of 1–4 μM. For the negative control 50 or 500 mM EDTA was added to the reaction mixture. The samples were filled into NT.LabelFree Standard Treated Capillaries (NanoTemper Technologies GmbH). The measurement took place at 22°C with laser off/on/off times of 5, 39, or 5 s. The instrument parameters were adjusted to 5% LED power and 20% MST power. The data presented here are from three technical replicates done in the same day; the whole experiment was repeated at least three times confirming the results. We analyzed the temperature jump (fluorescence change during the first second of IR radiation exposure) for each sample.

Fluorescence change during temperature jump was plotted against ligand concentration and curves were fitted with the Hill Fit:

f(c) = unbound + (bound - unbound)/(1 + (EC50/c)^{^}n)

in IGOR (Wavemetrics).

In order to identify interaction partners of otoferlin, we used E18 vestibular maculae of the chicken utricle, each containing more than 20000 hair cells for affinity purification of otoferlin. Most vestibular hair cells are functional at this late embryonic age (Goodyear et al., 1999), and utricles can be dissected relatively quickly in larger numbers (Herget et al., 2013). Membrane proteins of 60 avian utricular maculae were solubilized with octyl-β-D-glucopyranoside (Kim et al., 2004) and otoferlin and its potential binding partners were purified using the monoclonal anti-chicken otoferlin antibody HCS-1 (Goodyear et al., 2010), immobilized to magnetic dynabeads. The HCS-1 antibody binds strongly and specifically to chicken otoferlin, but does not recognize mammalian otoferlin.

Specificity of the otoferlin IP was assessed by comparison to a control pull-down using a monoclonal antibody to chicken tip-link antigen protocadherin 15 (Goodyear and Richardson, 2003; Kazmierczak et al., 2007). Eluates of both otoferlin and the control IPs were analyzed by LC-MS/MS. Otoferlin was specifically and efficiently immunoprecipitated with the HSC-1 antibody and 161 otoferlin peptides were identified covering 74% of the chicken utricle protein sequence (Supplementary Figure S1). No otoferlin peptides were found in the control pull-down.

In 11 out of 11 independent IPs, CaMKIIδ co-purified with otoferlin, with at least 4 peptides, 95% peptide threshold, and 99.9% protein probability. CaMKIIδ was not co-purified in the control IPs.

CaMKIIδ is an important modulator of synapses, but has not been described to play a role in the inner ear. We therefore aimed to find out whether CaMKIIδ interacts with otoferlin and phosphorylates it in mammalian auditory hair cells.

We investigated the localization and expression of all CaMKIIs in mammalian IHCs using immunohistochemistry (Figure 1) and PCR (Figure 2).

In mouse organs of Corti at P14 we found hardly any immunolabelling for CaMKIIα, CaMKIIβ or CaMKIIγ within the cytoplasm of IHCs, which were co-labeled for otoferlin (Figures 1A–C). Immunolabelling against CaMKIIβ, CaMKIIγ and CaMKIIδ appeared outside of IHCs, possibly in efferent and/or afferent synaptic boutons (Figures 1B–D). For CaMKIIδ, immunoreactivity could be detected also within the cytoplasm of IHCs (Figure 1D). To narrow down the localization of CaMKIIδ at the synapse, we co-labeled with the ribbon protein Ctbp2 and the postsynaptic marker PSD95. Immunolabelling revealed CaMKIIδ to localize close to PSD95, at the opposite side of the ribbon (Figure 1E), indicating a postsynaptic localization of CaMKIIδ in afferent synaptic boutons, in addition to the cytoplasmic localization within the IHCs.

To test for different CaMKII mRNA transcripts in IHCs, we designed PCR primers specific for each of the four CaMKII genes. Suitability to amplify the respective CaMKII transcripts was confirmed using mouse brain cDNA as template in real-time PCR with SYBR green (Figure 2E). 3–5 IHCs per sample were collected with a patch pipette and mRNA was reverse transcribed. Only samples displaying TaqMan-PCR signals for bassoon and TATA-binding protein as housekeeping genes were considered for analysis. Real-time PCR experiments revealed the presence of CaMKIIδ transcripts in three independent samples (Figures 2A–C). CaMKIIγ mRNA could be detected in one out of three samples (Figure 2C), while transcripts from CaMKIIα and CaMKIIβ could not be amplified in any IHC sample (Figure 2E). Therefore, we conclude that CaMKIIδ is the predominant CaMKII in rodent IHCs, with a supporting contribution by CaMKIIγ.

We next investigated whether CaMKIIδ and otoferlin interact in mammalian IHCs using an immunohistochemistry based in situ proximity ligation assay (PLA) (Figure 3) which detects a <40 nm proximity of antibody-labeled proteins (Koos et al., 2014).

First, we validated the PLA with a previously reported interaction of otoferlin with myosin VI (Figure 3; Heidrych et al., 2009; Roux et al., 2009). We applied the PLA in acutely isolated P11 rat organ of Corti explants resulting in discrete fluorescent puncta distributed over the whole IHC body (Figure 3B), indicating close proximity (<40 nm) of otoferlin and myosin VI, likely due to physical interaction. When the PLA assay was done with only one primary antibody to otoferlin as a control (Figure 3B, middle panel), no puncta were detected. Similarly, no PLA signals were detectable when we performed the PLA with antibodies to otoferlin and parvalbumin (Figure 3D and cartoon), another hair cell marker that – like myosin VI – labels the whole IHC body but is not described as otoferlin interaction partner (Figure 3C).

Next, we used the PLA to verify a molecular interaction of otoferlin with CaMKIIδ in rat IHCs in situ (Figure 4). A close proximity of both proteins was indicated by fluorescent puncta in IHCs, suggesting CaMKIIδ to be an otoferlin interaction partner in mammalian cochlear IHCs (Figure 4B). PLA puncta also appeared with the pan-CaMKII antibody (Figure 4D), which was raised against the kinase domain that is highly conserved between the four CaMKII genes. No PLA signals were detected in control assays, using anti-CaMKII antibodies only (Figures 4B,D, middle panels).

While a positive PLA signal could in principle result from an indirect protein interaction via scaffold proteins, we tested whether CaMKIIδ and otoferlin interact directly in vitro. We expressed two HA-tagged fragments of mouse otoferlin, one comprising the first three C₂ domains (C₂ABC-HA, 70 kDa), and one comprising the last three C₂ domains (C₂DEF-HA, 114 kDa) as well as full-length mcherry-tagged CaMKIIδ (84 kDa) in HEK293 cells and performed IPs with an anti-HA antibody. In western blots, we identified a ∼84 kDa CaMKIIδ-mcherry band in the eluate of the co-IPs indicating a co-purification with otoferlin C₂ABC (Figure 5A). A fainter band was detected in the eluate when CaMKIIδ was co-purified with C₂DEF (Figure 5B), suggesting a weaker interaction. To mimic an interaction with full-length otoferlin, we co-expressed CaMKIIδ-mcherry with both C₂ABC-HA and C₂DEF-HA fragments, resulting again in the co-precipitation of the ∼84 kDa band of CaMKIIδ-mcherry in the eluate (Figure 5C).

We next performed in vitro phosphorylation assays to assess whether CaMKIIδ phosphorylates recombinant otoferlin in a Ca²⁺/calmodulin-dependent manner. We used E. coli as an expression system because recombinant proteins produced in bacteria lack phosphorylation (Sahdev et al., 2008). We combined purified otoferlin fragments (C₂ABC and C₂DEF) with or without recombinant CaMKIIδ and activated the phosphorylation reaction with Ca²⁺ and calmodulin. After incubation for 5 min, otoferlin C₂ABC and C₂DEF fragments were analyzed for phosphorylation by LC-MS/MS after in-gel trypsinization (Figure 6A). Phosphorylation sites were identified by 80 Da mass shifts in the respective peptides (Supplementary Figure S2).

Only phosphorylation sites identified in three independent experiments were considered. We found that both the N-terminal otoferlin C₂ABC fragment as well as the C-terminal C₂DEF fragment were phosphorylated when incubated with CaMKIIδ in vitro and lacked phosphorylation in the absence of the kinase. In total, we identified ten phosphorylation sites (P1 to P10, Figure 6B). Five sites are located within C₂ domains of otoferlin, including T448 (C₂C domain), S985 (C₂D domain), and S1777, S1808, T1860 (C₂F domain; C₂ domain borders according to Jiménez and Bashir, 2007). S1184 and T1197 were identified in a region between the C₂D and the C₂E domains which has been hypothesized to fold as a C₂ domain (C₂de; Washington and Ward, 2006; Han and Campbell, 2007; Pangršič et al., 2012). Most of the identified phosphorylation sites were found to be conserved between mammalian and non-mammalian otoferlin orthologs (Figure 7), but only a few are conserved within C₂ domains of the ferlin protein family (Figure 8). Five out of the ten phosphorylation sites followed the CaMKII consensus sequence R/K-X-X-S/T (White et al., 1998). Noticeably, in six phosphopeptides a hydrophobic leucine was found at the P+1 site of the phosphoserine or threonine (indicated in blue in Figures 7, 8), displaying a preferred residue for CaMKII recognition (Stokoe et al., 1993).

According to the comparison between ferlin protein family members, the position of the phosphorylated threonine at P1 is occupied by negatively charged aspartate residues in dysferlin and myoferlin (Figure 8), which are positioned in the top loops of the C₂ domain, just next to aspartate residues predicted to coordinate Ca²⁺ (Jiménez and Bashir, 2007). We therefore addressed the influence of CaMKIIδ phosphorylation on the Ca²⁺ affinity of the recombinant otoferlin C₂C domain. To mimic the negative charge introduced by phosphorylation, we mutated threonine residue T448 into an aspartate residue (T448D). Ca²⁺ affinity was assessed by microscale thermophoresis (van den Bogaart et al., 2012) (Figure 9).

For the non-phosphorylated C₂C domain no changes in fluorescence were detected for Ca²⁺ concentrations between 6 μM and 200 mM compared to the negative control carried out in the presence of 500 mM EDTA (Figure 9A). Thus, either Ca²⁺ binding occurred but did not change the thermophoresis signal, or the non-phosphorylated C₂C domain did not bind to Ca²⁺ in this assay. Accordingly, the recombinant otoferlin C₂ABC construct revealed no change in thermophoresis signal, suggesting lack of Ca²⁺ binding (Figure 9A). For the phosphomimetic C₂C domain (T448D) we observed a fluorescence change above the one triggered by Ca²⁺ plus EDTA (Figure 9B). Curve fitting resulted in an apparent dissociation constant (K_Ca) of 8.7 ± 2.8 mM. Hence, phosphorylation by CaMKIIδ most likely converts the C₂C domain from a non-Ca²⁺ binding into a Ca²⁺ binding C₂ domain (Figure 9C), although with rather low Ca²⁺ affinity.

Next, we assessed the effect of phosphorylation on Ca²⁺ affinity of the C₂F domain. For the non-phosphorylated C₂F domain, we found an apparent dissociation constant for Ca²⁺ of 402 ± 54 μM, which was abolished in the presence of EDTA (Figure 9D). Using Mg²⁺ instead of Ca²⁺, we also detected a change in fluorescence, yet a Mg²⁺ concentration of 20 mM was not sufficient to reach a plateau, indicating a rather low Mg²⁺ affinity of the C₂F domain (Figure 9E). We then mimicked phosphorylation of P8, P9 and P10 by replacing the respective serine/threonine residues by aspartates (S1777D, S1808D, T1860D). The phosphomimetic C₂F domain binds Ca²⁺ with a K_Ca of 6.7 ± 0.7 mM (Figure 9F, corresponding negative control: Figure 9G). For Mg²⁺ we obtained an apparent dissociation constant of 16.2 ± 7.2 mM (Figure 9H), which is at least one order of magnitude above the intracellular concentration of free Mg²⁺ (<1 mM) (Romani and Scarpa, 1992). To assess the effect of salt concentration on the C₂F domain stability and hence Ca²⁺ affinity, we measured the K_Ca with 150 mM NaCl as above (Figure 9D) compared to 300 mM NaCl. As indicated in Figure 9I, the fluorescence changes reached a plateau earlier for high salt concentrations, resulting in a lower apparent K_{Ca(high salt)} of 330 ± 28 μM.

In summary, phosphomimetic mutations decreased the Ca²⁺ affinity of the C₂F domain by at least one order of magnitude. This suggests that phosphorylation by CaMKIIδ likely results in a lower Ca²⁺ affinity of the otoferlin C₂F domain.

Using a PLA to find phosphoserine residues in <40 nm distance from otoferlin, we next tested whether otoferlin and/or proteins interacting with otoferlin are phosphorylated in IHCs (Figure 10). To assess whether otoferlin phosphorylation depends on hair cell activity in vivo, we applied low or high external K⁺ solutions to acutely isolated OCs. High K⁺ depolarizes the plasma membrane leading to opening of voltage gated Ca²⁺ channels, and the Ca²⁺-influx triggers Ca²⁺-induced exocytosis but is also predicted to activate CaMKII by the Ca²⁺/calmodulin complex. We found PLA punctae in IHCs at resting conditions (Figure 10B), indicating a basal level of phosphorylated otoferlin or otoferlin interaction partners. After exposure to high K⁺, we observed more and brighter PLA signals (Figure 10B, middle panel). Comparing mean pixel intensities of the fluorescent PLA signals in resting IHCs with the ones in IHCs after stimulation, we found a four-fold fluorescence increase in high K⁺-stimulated IHCs (Figure 10C). This suggests a higher degree of otoferlin phosphorylation per otoferlin molecule, a higher number of phosphorylated otoferlin molecules, a higher degree of phosphorylation of proteins interacting with otoferlin, an increased interaction of otoferlin with a phosphorylated protein, or a combination of these possibilities.

To assess whether this increase in phosphorylation of otoferlin or the otoferlin interactome is mediated by CaMKII in rat IHCs, we pre-incubated acutely dissected OCs with the cell-permeable CaMKII inhibitor KN-93, which competitively interacts with the Ca²⁺/calmodulin-binding site on CaMKII, (Sumi et al., 1991). In the presence of KN-93, the stimulation-dependent increase in PLA signal was blocked to a large extent (Figures 10B,C), suggesting that activity-dependent phosphorylation of otoferlin or the otoferlin interactome in rat IHCs is indeed mostly due to the CaMKII kinase reaction.

Because of its presumed function in synaptic transmission, we next analyzed if phosphorylated otoferlin or the phosphorylated protein complex localizes to synaptic ribbons. We performed the PLA assay as before with 15 min high K⁺-stimulation and co-stained against the ribbon marker Ctbp2 (Figure 10D). PLA puncta, likely reflecting phosphorylated otoferlin and/or otoferlin bound to a phosphorylated protein, did not co-localize with the synaptic ribbons, but were rather found to be in the vicinity or adjacent to each other. This suggests that CaMKIIδ regulates otoferlin activity near the active zone membrane or in endocytotic compartments which are both in close proximity to the synaptic ribbon (Duncker et al., 2013; Neef et al., 2014; Revelo et al., 2014; Jung et al., 2015).

In summary, protein complexes containing otoferlin seem to be phosphorylated adjacent to synaptic ribbons in vivo. Phosphorylation is strongly promoted by hair cell stimulation and can be blocked by a CaMKII inhibitor.