Visualizing Collagen Fibrils in the Cochlea's Tectorial and Basilar Membranes Using a Fluorescently Labeled Collagen-Binding Protein Fragment.

PURPOSE: A probe that binds to unfixed collagen fibrils was used to image the shapes and fibrous properties of the TM and BM. The probe (CNA35) is derived from the bacterial adhesion protein CNA. We present confocal images of hydrated gerbil TM, BM, and other cochlear structures stained with fluorescently labeled CNA35. A primary purpose of this article is to describe the use of the CNA35 collagen probe in the cochlea. METHODS: Recombinant poly-histidine-tagged CNA35 was expressed in Escherichia coli, purified by cobalt-affinity chromatography, fluorescence labeled, and further purified by gel filtration chromatography. Cochleae from freshly harvested gerbil bullae were irrigated with and then incubated in CNA35 for periods ranging from 2 h - overnight. The cochleae were fixed, decalcified, and dissected. Isolated cochlear turns were imaged by confocal microscopy. RESULTS: The CNA35 probe stained the BM and TM, and volumetric imaging revealed the shape of these structures and the collagen fibrils within them. The limbal zone of the TM stained intensely. In samples from the cochlear base, intense staining was detected on the side of the TM that faces hair cells. In the BM pectinate zone, staining was intense at the upper and lower boundaries. The BM arcuate zone was characterized by a prominent longitudinal collagenous structure. The spiral ligament, limbus and lamina stained for collagen, and within the spiral limbus the habenula perforata were outlined with intense staining. CONCLUSION: The CNA35 probe provides a unique and useful view of collagenous structures in the cochlea.

Development of Ultrasensitive Biomimetic Auditory Hair Cells Based on Piezoresistive Hydrogel Nanocomposites.

With an ageing population, hearing disorders are predicted to rise considerably in the following decades. Thus, developing a new class of artificial auditory system has been highlighted as one of the most exciting research topics for biomedical applications. Herein, a design of a biocompatible piezoresistive-based artificial hair cell sensor is presented consisting of a highly flexible and conductive polyvinyl alcohol (PVA) nanocomposite with vertical graphene nanosheets (VGNs). The bilayer hydrogel sensor demonstrates excellent performance to mimic biological hair cells, responding to acoustic stimuli in the audible range between 60 Hz to 20 kHz. The sensor output demonstrates stable mid-frequency regions (∼4-9 kHz), with the greatest sensitivity as high frequencies (∼13-20 kHz). This is somewhat akin to the mammalian auditory system, which has remarkable sensitivity and sharp tuning at high frequencies due to the "active process". This work validates the PVA/VGN sensor as a potential candidate to play a similar functional role to that of the cochlear hair cells, which also operate over a wide frequency domain in a viscous environment. Further characterizations of the sensor show that increasing the sound amplitude results in higher responses from the sensor while taking it to the depth drops the sensor outputs due to attenuation of sound in water. Meanwhile, the acoustic pressure distribution of sound waves is predicted through finite element analysis, whereby the numerical results are in perfect agreement with experimental data. This proof-of-concept work creates a platform for the future design of susceptible, flexible biomimetic sensors to closely mimic the biological cochlea.

Mechanical forces drive ordered patterning of hair cells in the mammalian inner ear.

Periodic organization of cells is required for the function of many organs and tissues. The development of such periodic patterns is typically associated with mechanisms based on intercellular signaling such as lateral inhibition and Turing patterning. Here we show that the transition from disordered to ordered checkerboard-like pattern of hair cells and supporting cells in the mammalian hearing organ, the organ of Corti, is likely based on mechanical forces rather than signaling events. Using time-lapse imaging of mouse cochlear explants, we show that hair cells rearrange gradually into a checkerboard-like pattern through a tissue-wide shear motion that coordinates intercalation and delamination events. Using mechanical models of the tissue, we show that global shear and local repulsion forces on hair cells are sufficient to drive the transition from disordered to ordered cellular pattern. Our findings suggest that mechanical forces drive ordered hair cell patterning in a process strikingly analogous to the process of shear-induced crystallization in polymer and granular physics.

Stereocilia Rootlets: Actin-Based Structures That Are Essential for Structural Stability of the Hair Bundle.

Sensory hair cells of the inner ear rely on the hair bundle, a cluster of actin-filled stereocilia, to transduce auditory and vestibular stimuli into electrical impulses. Because they are long and thin projections, stereocilia are most prone to damage at the point where they insert into the hair cell's soma. Moreover, this is the site of stereocilia pivoting, the mechanical movement that induces transduction, which additionally weakens this area mechanically. To bolster this fragile area, hair cells construct a dense core called the rootlet at the base of each stereocilium, which extends down into the actin meshwork of the cuticular plate and firmly anchors the stereocilium. Rootlets are constructed with tightly packed actin filaments that extend from stereocilia actin filaments which are wrapped with TRIOBP; in addition, many other proteins contribute to the rootlet and its associated structures. Rootlets allow stereocilia to sustain innumerable deflections over their lifetimes and exemplify the unique manner in which sensory hair cells exploit actin and its associated proteins to carry out the function of mechanotransduction.

Single-cell proteomics reveals changes in expression during hair-cell development.

Hearing and balance rely on small sensory hair cells that reside in the inner ear. To explore dynamic changes in the abundant proteins present in differentiating hair cells, we used nanoliter-scale shotgun mass spectrometry of single cells, each ~1 picoliter, from utricles of embryonic day 15 chickens. We identified unique constellations of proteins or protein groups from presumptive hair cells and from progenitor cells. The single-cell proteomes enabled the de novo reconstruction of a developmental trajectory using protein expression levels, revealing proteins that greatly increased in expression during differentiation of hair cells (e.g., OCM, CRABP1, GPX2, AK1, GSTO1) and those that decreased during differentiation (e.g., TMSB4X, AGR3). Complementary single-cell transcriptome profiling showed corresponding changes in mRNA during maturation of hair cells. Single-cell proteomics data thus can be mined to reveal features of cellular development that may be missed with transcriptomics.

In Vivo Calcium Imaging of Lateral-line Hair Cells in Larval Zebrafish.

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory stimuli that mechanically deflect apical protrusions called hair bundles. Deflection opens mechanotransduction (MET) channels in hair bundles, leading to an influx of cations, including calcium. This cation influx depolarizes the cell and opens voltage-gated calcium channels located basally at the hair-cell presynapse. In mammals, hair cells are encased in bone, and it is challenging to functionally assess these activities in vivo. In contrast, larval zebrafish are transparent and possess an externally located lateral-line organ that contains hair cells. These hair cells are functionally and structurally similar to mammalian hair cells and can be functionally assessed in vivo. This article outlines a technique that utilizes a genetically encoded calcium indicator (GECI), GCaMP6s, to measure stimulus-evoked calcium signals in zebrafish lateral-line hair cells. GCaMP6s can be used, along with confocal imaging, to measure in vivo calcium signals at the apex and base of lateral-line hair cells. These signals provide a real-time, quantifiable readout of both mechanosensation- and presynapse-dependent calcium activities within these hair cells. These calcium signals also provide important functional information regarding how hair cells detect and transmit sensory stimuli. Overall, this technique generates useful data about relative changes in calcium activity in vivo. It is less well-suited for quantification of the absolute magnitude of calcium changes. This in vivo technique is sensitive to motion artifacts. A reasonable amount of practice and skill are required for proper positioning, immobilization, and stimulation of larvae. Ultimately, when properly executed, the protocol outlined in this article provides a powerful way to collect valuable information about the activity of hair-cells in their natural, fully integrated states within a live animal.

Homeostatic Control of Spontaneous Activity in the Developing Auditory System.

Neurons in the developing auditory system exhibit spontaneous bursts of activity before hearing onset. How this intrinsically generated activity influences development remains uncertain, because few mechanistic studies have been performed in vivo. We show using macroscopic calcium imaging in unanesthetized mice that neurons responsible for processing similar frequencies of sound exhibit highly synchronized activity throughout the auditory system during this critical phase of development. Spontaneous activity normally requires synaptic excitation of spiral ganglion neurons (SGNs). Unexpectedly, tonotopic spontaneous activity was preserved in a mouse model of deafness in which glutamate release from hair cells is abolished. SGNs in these mice exhibited enhanced excitability, enabling direct neuronal excitation by supporting cell-induced potassium transients. These results indicate that homeostatic mechanisms maintain spontaneous activity in the pre-hearing period, with significant implications for both circuit development and therapeutic approaches aimed at treating congenital forms of deafness arising through mutations in key sensory transduction components.

Structural relationship between the putative hair cell mechanotransduction channel TMC1 and TMEM16 proteins.

The hair cell mechanotransduction (MET) channel complex is essential for hearing, yet it's molecular identity and structure remain elusive. The transmembrane channel-like 1 (TMC1) protein localizes to the site of the MET channel, interacts with the tip-link responsible for mechanical gating, and genetic alterations in TMC1 alter MET channel properties and cause deafness, supporting the hypothesis that TMC1 forms the MET channel. We generated a model of TMC1 based on X-ray and cryo-EM structures of TMEM16 proteins, revealing the presence of a large cavity near the protein-lipid interface that also harbors the Beethoven mutation, suggesting that it could function as a permeation pathway. We also find that hair cells are permeable to 3 kDa dextrans, and that dextran permeation requires TMC1/2 proteins and functional MET channels, supporting the presence of a large permeation pathway and the hypothesis that TMC1 is a pore forming subunit of the MET channel complex.

Oncomodulin Expression Reveals New Insights into the Cellular Organization of the Murine Utricle Striola.

Oncomodulin (OCM, aka ß-parvalbumin) is an EF-hand calcium binding protein that is expressed in a restricted set of hair cells in the peristriolar region of the mammalian utricle. In the present study, we determined the topologic distribution of OCM among hair cell phenotypes to advance our understanding of the cellular organization of the striola and the relationship of these phenotypes with characteristics of tissue polarity. The distributions of OCM-positive (OCM+) hair cells were quantified in utricles of mature C57Bl/6 mice. Immunohistochemistry was conducted using antibodies to OCM, calretinin, and ß3-tubulin. Fluorophore-conjugated phalloidin was used to label hair cell stereocilia, which provided the basis for determining hair cell counts and morphologic polarizations. We found OCM expression in striolar types I and II hair cells, though the distributions were dissimilar to the native striolar type I and II distributions, favoring type I hair cells. The distribution of OCM immunoreactivity among striolar type I hair cells also reflected nonrandom distribution among type Ic and Id phenotypes (i.e., those receiving calretinin-positive and calretinin-negative calyces, respectively). However, many OCM+ hair cells were found lateral to the striola, and within the epithelial region encompassing OCM+ hair cells, the distributions of OCM+ types Ic and Id hair cells were similar to the native distributions of Ic and Id in this region. Summarily, these data provide a quantitative perspective supporting the existence of different underlying factors driving the topologic expression of OCM in hair cells than those responsible for tissue polarity characteristics associated within the utricular striola, including calretinin expression in afferent calyces.

Phosphoinositol-4,5-Bisphosphate Regulates Auditory Hair-Cell Mechanotransduction-Channel Pore Properties and Fast Adaptation.

Membrane proteins, such as ion channels, interact dynamically with their lipid environment. Phosphoinositol-4,5-bisphosphate (PIP2) can directly or indirectly modify ion-channel properties. In auditory sensory hair cells of rats (Sprague Dawley) of either sex, PIP2 localizes within stereocilia, near stereocilia tips. Modulating the amount of free PIP2 in inner hair-cell stereocilia resulted in the following: (1) the loss of a fast component of mechanoelectric-transduction current adaptation, (2) an increase in the number of channels open at the hair bundle's resting position, (3) a reduction of single-channel conductance, (4) a change in ion selectivity, and (5) a reduction in calcium pore blocking effects. These changes occur without altering hair-bundle compliance or the number of functional stereocilia within a given hair bundle. Although the specific molecular mechanism for PIP2 action remains to be uncovered, data support a hypothesis for PIP2 directly regulating channel conformation to alter calcium permeation and single-channel conductance.SIGNIFICANCE STATEMENT How forces are relayed to the auditory mechanoelectrical transduction (MET) channel remains unknown. However, researchers have surmised that lipids might be involved. Previous work on bullfrog hair cells showed an effect of phosphoinositol-4,5-bisphosphate (PIP2) depletion on MET current amplitude and adaptation, leading to the postulation of the existence of an underlying myosin-based adaptation mechanism. We find similar results in rat cochlea hair cells but extend these data to include single-channel analysis, hair-bundle mechanics, and channel-permeation properties. These additional data attribute PIP2 effects to actions on MET-channel properties and not motor interactions. Further findings support PIP2's role in modulating a fast, myosin-independent, and Ca2+-independent adaptation process, validating fast adaptation's biological origin. Together this shows PIP2's pivotal role in auditory MET, likely as a direct channel modulator.

Functional Analysis of the Transmembrane and Cytoplasmic Domains of Pcdh15a in Zebrafish Hair Cells.

Protocadherin 15 (PCDH15) is required for mechanotransduction in sensory hair cells as a component of the tip link. Isoforms of PCDH15 differ in their cytoplasmic domains (CD1, CD2, and CD3), but share the extracellular and transmembrane (TMD) domains, as well as an intracellular domain known as the common region (CR). In heterologous expression systems, both the TMD and CR of PCDH15 have been shown to interact with members of the mechanotransduction complex. The in vivo significance of these protein-protein interaction domains of PCDH15 in hair cells has not been determined. Here, we examined the localization and function of the two isoforms of zebrafish Pcdh15a (CD1 and CD3) in pcdh15a-null mutants by assessing Pcdh15a transgene-mediated rescue of auditory/vestibular behavior and hair cell morphology and activity. We found that either isoform alone was able to rescue the Pcdh15a-null phenotype and that the CD1- or CD3-specific regions were dispensable for hair bundle integrity and labeling of hair cells with FM4-64, which was used as a proxy for mechanotransduction. When either the CR or TMD domain was deleted, the mutated proteins localized to the stereocilial tips, but were unable to rescue FM4-64 labeling. Disrupting both domains led to a complete failure of Pcdh15a to localize to the hair bundle. Our findings demonstrate that the TMD and cytoplasmic CR domains are required for the in vivo function of Pcdh15a in zebrafish hair cells.SIGNIFICANCE STATEMENT Tip links transmit force to mechanotransduction channels at the tip of hair bundles in sensory hair cells. One component of tip links is Protocadherin 15 (PCDH15). Here, we demonstrate that, when transgenically expressed, either zebrafish Pcdh15a-cytodomain 1 (CD1) or Pcdh15a-CD3 can rescue the phenotype of a pcdh15a-null mutant. Even when lacking the specific regions for CD1 or CD3, truncated Pcdh15a that contains the so-called common region (CR) at the cytoplasmic/membrane interface still has the ability to rescue similar to full-length Pcdh15a. In contrast, Pcdh15a lacking the entire cytoplasmic domain is not functional. These results demonstrate that the CR plays a key role in the mechanotransduction complex in hair cells.

ACF7 is a hair-bundle antecedent, positioned to integrate cuticular plate actin and somatic tubulin.

The precise morphology of the mechanosensitive hair bundle requires seamless integration of actin and microtubule networks. Here, we identify Acf7a (actin crosslinking family protein 7a) as a protein positioned to bridge these distinct cytoskeletal networks in hair cells. By imaging Acf7a-Citrine fusion protein in zebrafish and immunolabeling of vestibular and cochlear mouse hair cells, we show that Acf7a and ACF7 circumscribe, underlie, and are interwoven into the cuticular plate (CP), and they also encircle the basal body of the kinocilium. In cochlear hair cells, ACF7 localization is graded, with the highest concentration near each fonticulus--an area free of F-actin in the region of the CP that contains the basal body. During hair-cell development and regeneration, Acf7a precedes formation of the hair bundle and CP. Finally, electron tomography demonstrates that the ends of microtubules insert into the CP and are decorated with filamentous linkers connecting microtubules to the CP. These observations are consistent with ACF7 being a linker protein, which may shape the cytoskeleton of the hair cell early during hair-bundle genesis.

Permeation properties of the hair cell mechanotransducer channel provide insight into its molecular structure.

Mechanoelectric transducer (MET) channels, located near stereocilia tips, are opened by deflecting the hair bundle of sensory hair cells. Defects in this process result in deafness. Despite this critical function, the molecular identity of MET channels remains a mystery. Inherent channel properties, particularly those associated with permeation, provide the backbone for the molecular identification of ion channels. Here, a novel channel rectification mechanism is identified, resulting in a reduced pore size at positive potentials. The apparent difference in pore dimensions results from Ca(2+) binding within the pore, occluding permeation. Driving force for permeation at hyperpolarized potentials is increased because Ca(2+) can more easily be removed from binding within the pore due to the presence of an electronegative external vestibule that dehydrates and concentrates permeating ions. Alterations in Ca(2+) binding may underlie tonotopic and Ca(2+)-dependent variations in channel conductance. This Ca(2+)-dependent rectification provides targets for identifying the molecular components of the MET channel.

Whirlin interacts with espin and modulates its actin-regulatory function: an insight into the mechanism of Usher syndrome type II.

Whirlin mutations cause retinal degeneration and hearing loss in Usher syndrome type II (USH2) and non-syndromic deafness, DFNB31. Its protein recruits other USH2 causative proteins to form a complex at the periciliary membrane complex in photoreceptors and the ankle link of the stereocilia in hair cells. However, the biological function of this USH2 protein complex is largely unknown. Using a yeast two-hybrid screen, we identified espin, an actin-binding/bundling protein involved in human deafness when defective, as a whirlin-interacting protein. The interaction between these two proteins was confirmed by their coimmunoprecipitation and colocalization in cultured cells. This interaction involves multiple domains of both proteins and only occurs when espin does not bind to actin. Espin was partially colocalized with whirlin in the retina and the inner ear. In whirlin knockout mice, espin expression changed significantly in these two tissues. Further studies found that whirlin increased the mobility of espin and actin at the actin bundles cross-linked by espin and, eventually, affected the dimension of these actin bundles. In whirlin knockout mice, the stereocilia were thickened in inner hair cells. We conclude that the interaction between whirlin and espin and the balance between their expressions are required to maintain the actin bundle network in photoreceptors and hair cells. Disruption of this actin bundle network contributes to the pathogenic mechanism of hearing loss and retinal degeneration caused by whirlin and espin mutations. Espin is a component of the USH2 protein complex and could be a candidate gene for Usher syndrome.

Cholesterol influences voltage-gated calcium channels and BK-type potassium channels in auditory hair cells.

The influence of membrane cholesterol content on a variety of ion channel conductances in numerous cell models has been shown, but studies exploring its role in auditory hair cell physiology are scarce. Recent evidence shows that cholesterol depletion affects outer hair cell electromotility and the voltage-gated potassium currents underlying tall hair cell development, but the effects of cholesterol on the major ionic currents governing auditory hair cell excitability are unknown. We investigated the effects of a cholesterol-depleting agent (methyl beta cyclodextrin, MßCD) on ion channels necessary for the early stages of sound processing. Large-conductance BK-type potassium channels underlie temporal processing and open in a voltage- and calcium-dependent manner. Voltage-gated calcium channels (VGCCs) are responsible for calcium-dependent exocytosis and synaptic transmission to the auditory nerve. Our results demonstrate that cholesterol depletion reduced peak steady-state calcium-sensitive (BK-type) potassium current by 50% in chick cochlear hair cells. In contrast, MßCD treatment increased peak inward calcium current (~30%), ruling out loss of calcium channel expression or function as a cause of reduced calcium-sensitive outward current. Changes in maximal conductance indicated a direct impact of cholesterol on channel number or unitary conductance. Immunoblotting following sucrose-gradient ultracentrifugation revealed BK expression in cholesterol-enriched microdomains. Both direct impacts of cholesterol on channel biophysics, as well as channel localization in the membrane, may contribute to the influence of cholesterol on hair cell physiology. Our results reveal a new role for cholesterol in the regulation of auditory calcium and calcium-activated potassium channels and add to the growing evidence that cholesterol is a key determinant in auditory physiology.

The crystal structure of the C2A domain of otoferlin reveals an unconventional top loop region.

Otoferlin (Otof), whose genetic mutations cause profound deafness in humans, is a protein composed of at least six C(2) domains, which are known as Ca(2)(+)-binding and phospholipid-binding regions. Mammalian ferlin proteins are proposed to act in membrane fusion events, with Otof being specifically required for exocytosis in auditory hair cells. Ferlin C(2) domains exhibit a rather low level of sequence similarity to those of synaptotagmins, protein kinase C isoforms, or phospholipases. Here, we report the crystal structure of the N-terminal C(2) domain of Otof (C2A) at 1.95-Å resolution. In contrast to previous predictions, we found that this C(2) domain is complete with eight ß-strands. Comparing the structure of Otof C2A to those of other C(2) domains revealed one top loop in Otof to be significantly shorter. This results in a depression of the surface, which is positively charged for the Otof C2A domain, and contrasts with the head-like protrusion surrounded by a negatively charged "neck" typically found in other C(2) domains. Isothermal titration calorimetry and circular dichroism spectroscopy studies confirmed that Otof C2A is unable to bind Ca(2+), while the synaptotagmin-1 C2A domain exhibited Ca(2+) binding under the same conditions. Furthermore, floatation assays revealed a failure of Otof C(2)A to bind to phospholipid membranes. Accordingly, no positively charged ß-groove-like surface structure, which is known to bind phosphatidylinositol-4,5-bisphosphate in other C(2) domains, was found at the respective position in Otof C2A. Taken together, these data demonstrate that the Otof C2A domain differs structurally and functionally from other C(2) domains.

Molecular anatomy of the hair cell's ribbon synapse.

Hearing depends on reliable and temporally precise neurotransmission by cochlear hair cells. The wide dynamic range and high sensitivity with which these cells encode acoustic stimuli are associated with a presynaptic specialization termed the presynaptic dense body or synaptic ribbon. Apposed to the presynaptic density, this spherical or flattened structure tethers a layer of synaptic vesicles and is thought to facilitate their exocytotic fusion. Although defining the molecular constituents of the hair cell's synaptic ribbon should contribute to our understanding of neurotransmitter release at this synapse, accomplishing this task has been slowed by the difficulty of obtaining sufficient amounts of starting material for protein analysis from hair cells. We isolated synaptic material from chicken cochleas, purified synaptic ribbons with specific immunological reagents, and identified the associated proteins by tandem mass spectrometry. Purification of the ribbons revealed a predominant composition of C-terminal-binding proteins, especially ribeye, in association with the small GTPase Rab3, which is possibly involved in attaching vesicles to the ribbon. In comparison with the components of conventional synapses and of retinal ribbon synapses, we observed that certain regulatory proteins are excluded from the hair cell's synapse. Using antisera against several of the novel proteins and membrane-trafficking components that we had identified, we documented their localization in isolated hair cells. Our results indicate that the ribbon synapses of hair cells display modifications to the presynaptic machinery that are associated with the high-fidelity transmission of acoustic signals to the brain.

The novel PMCA2 pump mutation Tommy impairs cytosolic calcium clearance in hair cells and links to deafness in mice.

The mechanotransduction process in hair cells in the inner ear is associated with the influx of calcium from the endolymph. Calcium is exported back to the endolymph via the splice variant w/a of the PMCA2 of the stereocilia membrane. To further investigate the role of the pump, we have identified and characterized a novel ENU-induced mouse mutation, Tommy, in the PMCA2 gene. The mutation causes a non-conservative E629K change in the second intracellular loop of the pump that harbors the active site. Tommy mice show profound hearing impairment from P18, with significant differences in hearing thresholds between wild type and heterozygotes. Expression of mutant PMCA2 in CHO cells shows calcium extrusion impairment; specifically, the long term, non-stimulated calcium extrusion activity of the pump is inhibited. Calcium extrusion was investigated directly in neonatal organotypic cultures of the utricle sensory epithelium in Tommy mice. Confocal imaging combined with flash photolysis of caged calcium showed impairment of calcium export in both Tommy heterozygotes and homozygotes. Immunofluorescence studies of the organ of Corti in homozygous Tommy mice showed a progressive base to apex degeneration of hair cells after P40. Our results on the Tommy mutation along with previously observed interactions between cadherin-23 and PMCA2 mutations in mouse and humans underline the importance of maintaining the appropriate calcium concentrations in the endolymph to control the rigidity of cadherin and ensure the function of interstereocilia links, including tip links, of the stereocilia bundle.

A claudin-9-based ion permeability barrier is essential for hearing.

Hereditary hearing loss is one of the most common birth defects, yet the majority of genes required for audition is thought to remain unidentified. Ethylnitrosourea (ENU)-mutagenesis has been a valuable approach for generating new animal models of deafness and discovering previously unrecognized gene functions. Here we report on the characterization of a new ENU-induced mouse mutant (nmf329) that exhibits recessively inherited deafness. We found a widespread loss of sensory hair cells in the hearing organs of nmf329 mice after the second week of life. Positional cloning revealed that the nmf329 strain carries a missense mutation in the claudin-9 gene, which encodes a tight junction protein with unknown biological function. In an epithelial cell line, heterologous expression of wild-type claudin-9 reduced the paracellular permeability to Na+ and K+, and the nmf329 mutation eliminated this ion barrier function without affecting the plasma membrane localization of claudin-9. In the nmf329 mouse line, the perilymphatic K+ concentration was found to be elevated, suggesting that the cochlear tight junctions were dysfunctional. Furthermore, the hair-cell loss in the claudin-9-defective cochlea was rescued in vitro when the explanted hearing organs were cultured in a low-K+ milieu and in vivo when the endocochlear K+-driving force was diminished by deletion of the pou3f4 gene. Overall, our data indicate that claudin-9 is required for the preservation of sensory cells in the hearing organ because claudin-9-defective tight junctions fail to shield the basolateral side of hair cells from the K+-rich endolymph. In the tight-junction complexes of hair cells, claudin-9 is localized specifically to a subdomain that is underneath more apical tight-junction strands formed by other claudins. Thus, the analysis of claudin-9 mutant mice suggests that even the deeper (subapical) tight-junction strands have biologically important ion barrier function.