Tip Links Research Articles

A Large-Conductance Calcium-Selective Mechanotransducer Channel in Mammalian Cochlear Hair Cells

Sound stimuli are detected in the cochlea by opening of hair cell mechanotransducer (MT) channels, one of the few ion channels not yet conclusively identified at a molecular level. To define their performance in situ, we measured MT channel properties in inner hair cells (IHCs) and outer hair cells (OHCs) at two locations in the rat cochlea tuned to different characteristic frequencies (CFs). The conductance (in 0.02 mM calcium) of MT channels from IHCs was estimated as 260 pS at both low-frequency and mid-frequency positions, whereas that from OHCs increased with CFs from 145 to 210 pS. The combination of MT channel conductance and tip link number, assayed from scanning electron micrographs, accounts for variation in whole-cell current amplitude for OHCs and its invariance for IHCs. Channels from apical IHCs and OHCs having a twofold difference in unitary conductance were both highly calcium selective but were distinguishable by a small but significant difference in calcium permeability and in their response to lowering ionic strength. The results imply that the MT channel has properties possessed by few known candidates, and its diversity suggests expression of multiple isoforms.

Stereociliary Myosin-1c Receptors Are Sensitive to Calcium Chelation and Absent from Cadherin 23 Mutant Mice

The identities of some of the constituents of the hair-cell transduction apparatus have been elucidated only recently. The molecular motor myosin-1c (Myo1c) functions in adaptation of the hair-cell response to sustained mechanical stimuli and is therefore an integral part of the transduction complex. Recent data indicate that Myo1c interacts in vitro with two other molecules proposed to be important for transduction: cadherin 23 (Cdh23), a candidate for the stereociliary tip link, and phosphatidylinositol 4,5-bisphosphate (PIP2), which is abundant in the membranes of hair-cell stereocilia. It is not known, however, whether these interactions occur in hair cells. Using an in situ binding assay on saccular hair cells, we demonstrated previously that Myo1c interacts with molecules at stereociliary tips, the site of transduction, through sequences contained within its calmodulin (CaM)-binding neck domain, which can bind up to four CaM molecules. In the current study, we identify the second CaM-binding IQ domain as a region of Myo1c that mediates CaM-sensitive binding to stereociliary tips and to PIP2 immobilized on a solid support. Binding of Myo1c to stereociliary tips of cochlear and vestibular hair cells is disrupted by treatments that break tip links. In addition, Myo1c does not bind to stereocilia from mice whose hair cells lack Cdh23 protein despite the presence of PIP2 in the stereociliary membranes. Collectively, our data suggest that Myo1c and Cdh23 interact at the tips of hair-cell stereocilia and that this interaction is modulated by CaM.

The Tip-Link Antigen, a Protein Associated with the Transduction Complex of Sensory Hair Cells, Is Protocadherin-15

Sound and acceleration are detected by hair bundles, mechanosensory structures located at the apical pole of hair cells in the inner ear. The different elements of the hair bundle, the stereocilia and a kinocilium, are interconnected by a variety of link types. One of these links, the tip link, connects the top of a shorter stereocilium with the lateral membrane of an adjacent taller stereocilium and may gate the mechanotransducer channel of the hair cell. Mass spectrometric and Western blot analyses identify the tip-link antigen, a hitherto unidentified antigen specifically associated with the tip and kinocilial links of sensory hair bundles in the inner ear and the ciliary calyx of photoreceptors in the eye, as an avian ortholog of human protocadherin-15, a product of the gene for the deaf/blindness Usher syndrome type 1F/DFNB23 locus. Multiple protocadherin-15 transcripts are shown to be expressed in the mouse inner ear, and these define four major isoform classes, two with entirely novel, previously unidentified cytoplasmic domains. Antibodies to the three cytoplasmic domain-containing isoform classes reveal that each has a different spatiotemporal expression pattern in the developing and mature inner ear. Two isoforms are distributed in a manner compatible for association with the tip-link complex. An isoform located at the tips of stereocilia is sensitive to calcium chelation and proteolysis with subtilisin and reappears at the tips of stereocilia as transduction recovers after the removal of calcium chelators. Protocadherin-15 is therefore associated with the tip-link complex and may be an integral component of this structure and/or required for its formation.

The Very Large G-Protein-Coupled Receptor VLGR1: A Component of the Ankle Link Complex Required for the Normal Development of Auditory Hair Bundles

Sensory hair bundles in the inner ear are composed of stereocilia that can be interconnected by a variety of different link types, including tip links, horizontal top connectors, shaft connectors, and ankle links. The ankle link antigen is an epitope specifically associated with ankle links and the calycal processes of photoreceptors in chicks. Mass spectrometry and immunoblotting were used to identify this antigen as the avian ortholog of the very large G-protein-coupled receptor VLGR1, the product of the Usher syndrome USH2C (Mass1) locus. Like ankle links, Vlgr1 is expressed transiently around the base of developing hair bundles in mice. Ankle links fail to form in the cochleae of mice carrying a targeted mutation in Vlgr1 (Vlgr1/del7TM), and the bundles become disorganized just after birth. FM1-43 [N-(3-triethylammonium)propyl)-4-(4-(dibutylamino)styryl) pyridinium dibromide] dye loading and whole-cell recordings indicate mechanotransduction is impaired in cochlear, but not vestibular, hair cells of early postnatal Vlgr1/del7TM mutant mice. Auditory brainstem recordings and distortion product measurements indicate that these mice are severely deaf by the third week of life. Hair cells from the basal half of the cochlea are lost in 2-month-old Vlgr1/del7TM mice, and retinal function is mildly abnormal in aged mutants. Our results indicate that Vlgr1 is required for formation of the ankle link complex and the normal development of cochlear hair bundles.

Mechanical Properties and Consequences of Stereocilia and Extracellular Links in Vestibular Hair Bundles

Although knowledge of the fine structure of vestibular hair bundles is increasing, the mechanical properties and functional significance of those structures remain unclear. In 2004, Bashtanov and colleagues reported the contribution of different extracellular links to bundle stiffness. We simulated Bashtanov’s experimental protocol using a three-dimensional finite element bundle model with geometry measured from a typical striolar hair cell. Unlike any previous models, we separately consider two types of horizontal links: shaft links and upper lateral links. Our most important results are as follows. First, we identified the material properties required to match Bashtanov’s experiment: stereocilia Young’s modulus of 0.74 GPa, tip link assembly (gating spring) stiffness of 5300 pN/ μm, and the combined stiffness of shaft links binding two adjacent stereocilia of 750 ∼ 2250 pN/ μm. Second, we conclude that upper lateral links are likely to have nonlinear mechanical properties: they have minimal stiffness during small bundle deformations but stiffen as the bundle deflects further. Third, we estimated the stiffness of the gating spring based on our realistic three-dimensional bundle model rather than a conventional model relying on the parallel arrangement assumption. Our predicted stiffness of the gating spring was greater than the previous estimation.

Analysis of the Dynamic Behavior of the Inner Hair Cell Stereocilia by the Finite Element Method

The motion of the inner hair cell (IHC) stereocilia, which results in tension in the tip links connected to mechanically gated ion channels, mediates the auditory transduction process. However, it is difficult to directly observe the motion of the stereocilia because of their minute dimensions and complex structure. In this study, to investigate such motion, a finite element method model of the tall, middle and short IHC stereocilia, including the tip and lateral links extending between the stereocilia, was constructed. By applying an analytically estimated fluid force caused by a stimulus of 60dB SPL at 500Hz to the model, the dynamic behavior of the stereocilia was analyzed. Numerical results showed that the stereocilia moved in phase and that the maximum tensions of 2.5fN and 2.1fN occurred in the tip link connecting the tall and middle stereocilia and in the tip link connecting the middle and short stereocilia, respectively. By contrast, under the condition in which the lateral links were removed, maximum tension in the former increased to 11.6fN, while that in the latter only increased to 2.3fN. It was therefore suggested that the lateral links protect the MET channels located at taller stereocilia against large stimuli and subject the channels located in the same IHC to forces of similar size.

A model of peripherally developing hearing loss and tinnitus based on the role of hypoxia and ischemia

The incidence of sensorineural hearing loss often caused by direct damage to the cochlear hair cells is by far more frequent and more serious than disorders affecting the external ear or the middle ear. Mechanisms that are discussed to be relevant for the genesis of tinnitus and acquired hearing impairment are hair cell loss, signal transduction disturbances in the region of the outer and inner hair cells and the spiral ganglion, impairment of cochlear blood flow, mechanical disturbance, and hypoxia and ischemia. The present model surveys the possible cellular and molecular biological causes of peripherally developing hearing loss and tinnitus. In particular, the paper discusses the roles of hypoxia and ischemia in the cochlea and in the etiology of the neurosensory types of tinnitus. Peripheral origins of hearing disturbances and tinnitus may be: (a) damage to the stereocilia and the tip links, (b) dysfunction of potassium channels or (c) modification of the glutamate release. Moreover, the hypoxia inducible factor-1 may have an important role to play as a key transcription factor in the cells' adaptation to hypoxia and ischemia. An impairment of the cochlear blood flow may be induced by the expression of target genes like nitrogen monoxide synthase and endothelin-1 resulting in tinnitus. The paper discusses consequences resulting from the present model for the medical treatment of peripherally developing tinnitus and hearing loss.

Ca 2+ Changes the Force Sensitivity of the Hair-Cell Transduction Channel

The mechanically gated transduction channels of vertebrate hair cells tend to close in ∼1 ms after their activation by hair bundle deflection. This fast adaptation is correlated with a quick negative movement of the bundle (a “twitch”), which can exert force and may mediate an active mechanical amplification of sound stimuli in hearing organs. We used an optical trap to deflect bullfrog hair bundles and to measure bundle movement while controlling Ca 2+ entry with a voltage clamp. The twitch elicited by repolarization of the cell varied with force applied to the bundle, going to zero where channels were all open or closed. The force dependence is quantitatively consistent with a model in which a Ca 2+-bound channel requires ∼3 pN more force to open, and rules out other models for the site of Ca 2+ action. In addition, we characterized a faster, voltage-dependent “flick”, which requires intact tip links but not current through transduction channels.

Effect of fluid forcing on vestibular hair bundles

A dynamic 3-D hair bundle model including inertia and viscous fluid drag effects based on the finite element method is presented. Six structural components are used to construct the hair bundle--kinocilium, stereocilia, upper lateral links, shaft links, tip links, and kinocilial links. Fluid drag is distributed on the surface of cilia columns. Bundle mechanics are analyzed under two distinct loading conditions: (1) drag caused by the shear flow of the surrounding endolymph fluid (fluid-forced), (2) a single force applied to the tip of the kinocilium (point-forced). A striolar and a medial extrastriolar vestibular hair cell from the utricle of a turtle are simulated. The striolar cell bundle shows a clear difference in tip link tension profile between fluid-forced and point-forced cases. When the striolar cell is fluid forced, it shows more evenly distributed tip link tensions and is far more sensitive, responding like an on/off switch. The extrastriolar cell does not show noticeable differences between the forcing types. For both forcing conditions, the extrastriolar cell responds serially--the nearest tip links to the kinocilium get tensed first, then the tension propagates to the farther tip links.

BAPTA Induces Frequency Shifts in vivo of Spontaneous Otoacoustic Emissions of the Bobtail Lizard

Spontaneous otoacoustic emissions (SOAEs) are indicators of active processes in the inner ear and are found in all classes of land vertebrates. In the Australian bobtail lizard, earlier work showed that otoacoustic emissions are generated by an active motility process in the hair-cell bundle. This is likely to be driven by calcium-sensitive mechanisms implicated in other non-mammalian hair cell systems. If so, it should be fundamentally influenced by the extracellular calcium concentration. In in vitro studies, the rate of force generation in hair cell stereovilli is linked to the extracellular calcium concentration. In such preparations, low-calcium solutions, buffered by the calcium chelator BAPTA, were reported to change the frequency of hair cell bundle oscillations. In the present study, BAPTA was iontophoresed into the endolymph of the bobtail skink in vivo, and SOAEs were monitored. Application of BAPTA resulted in a prolonged downward shift in the frequency of individual SOAE spectral peaks. Recovery took more than 1 h, consistent with a slow clearance of BAPTA from endolymph. SOAE peak amplitudes were most often enhanced, suggesting there was no functional disruption of tip links. The direction and degree of frequency shifts were consistent with in vitroand in vivo data showing the effects of changing calcium concentrations in the endolymph directly.

Force on inner hair cell cilia

The cochlea of the inner ear transforms the incoming sound pressure into neural excitation. Despite extensive experiments and modeling for the past century, understanding the behavior of the cochlea is far from complete. With an efficient program (Fast4) for shell of revolution structures, all mechanical (elastic) details of the curved cochlear cross-section and the organ of Corti can be computed. Based on the known values for the elastic moduli of the protein fibers and estimates for the geometry, the responses to point and pressure loads have been calculated which are reasonably close to the direct measurements. In the present work, the details of the inner hair cell are included, with a fluid gap between the tip of the cilia and the Hensen stripe of the tectorial membrane. A simple model for the near contact indicates nonlinear response similar to the intracellular recordings. This near contact is included into a more complete elastic model for the organ of Corti that includes three rows of cilia and tip links. The phase of the maximum tension of the tip link, which causes excitation of the cell, is computed for low frequencies for comparison to measurements. For low frequencies the fluid motion in the organ of Corti is approximately two-dimensional. The phase is found to be affected by: (1) geometrical difference between basal and upper turns of the cochlea, (2) initial gap spacing between the tip of the cilium and the Hensen stripe, (3) initial gap spacing between the tip of the cilium and the tectorial membrane, (4) presence of an electrode probe constraint on the motion of the inner hair cell, and (5) the stiffness of the tectorial membrane. The latter is the most significant. For a soft tectorial membrane, the excitation is generally between maximum velocity and displacement of the basilar membrane toward scala vestibuli. However, for a stiff tectorial membrane, the phase changes to an excitation with velocity and displacement toward scala tympani. Thus a possible mechanical reason is offered for the auditory nerve excitation in the base of the chinchilla cochlea for basilar membrane velocity toward scala tympani and in the middle and upper turns of the guinea pig cochlea, excitation for velocity toward scala vestibuli.

In Search of the Hair-Cell Gating Spring

Mechanotransduction in vertebrate hair cells involves a biophysically defined elastic element (the "gating spring") that pulls on the transduction channels. The tip link, a fine filament made of cadherin 23 linking adjacent stereocilia in hair-cell bundles, has been suggested to be the gating spring. However, TRP channels that mediate mechanotransduction in Drosophila, zebrafish, and mice often have cytoplasmic domains containing a large number of ankyrin repeats that are also candidates for the gating spring. We have explored the elastic properties of cadherin and ankyrin repeats through molecular dynamics simulations using crystallographic structures of proteins with one cadherin repeat or 4 and 12 ankyrin repeats, and using models of 17 and 24 ankyrin repeats. The extension and stiffness of large ankyrin-repeat structures were found to match those predicted by the gating-spring model. Our results suggest that ankyrin repeats of TRPA1 and TRPN1 channels serve as the gating spring for mechanotransduction.

Development and properties of stereociliary link types in hair cells of the mouse cochlea

The hair bundles of outer hair cells in the mature mouse cochlea possess three distinct cell-surface specializations: tip links, horizontal top connectors, and tectorial membrane attachment crowns. Electron microscopy was used to study the appearance and maturation of these link types and examine additional structures transiently associated with the developing hair bundle. At embryonic day 17.5 (E17.5), the stereocilia are interconnected by fine lateral links and have punctate elements distributed over their surface. Oblique tip links are also seen at this stage. By postnatal day 2 (P2), outer hair cell bundles have a dense cell coat, but have lost many of the lateral links seen at E17.5. At P2, ankle links appear around the base of the bundle and tectorial membrane attachment crowns are seen at the stereociliary tips. Ankle links become less apparent by P9 and are completely lost by P12. The appearance of horizontal top connectors, which persist into adulthood, occurs concomitant with this loss of ankle links. Treatment with the calcium chelator BAPTA or the protease subtilisin enabled these links to be further distinguished. Ankle links are susceptible to both treatments, tip links are only sensitive to BAPTA, and tectorial membrane attachment crowns are removed by subtilisin but not BAPTA. The cell-coat material is partially sensitive to subtilisin alone, while horizontal top connectors resist both treatments. These results indicate there is a rich, rapidly changing array of different links covering the developing hair bundle that becomes progressively refined to generate the mature complement by P19.

Cadherin 23 is a component of the transient lateral links in the developing hair bundles of cochlear sensory cells

Cadherin 23 is required for normal development of the sensory hair bundle, and recent evidence suggests it is a component of the tip links, filamentous structures thought to gate the hair cells' mechano-electrical transducer channels. Antibodies against unique peptide epitopes were used to study the properties of cadherin 23 and its spatio-temporal expression patterns in developing cochlear hair cells. In the rat, intra- and extracellular domain epitopes are readily detected in the developing hair bundle between E18 and P5, and become progressively restricted to the distal tip of the hair bundle. From P13 onwards, these epitopes are no longer detected in hair bundles, but immunoreactivity is observed in the apical, vesicle-rich, pericuticular region of the hair cell. In the P2–P3 mouse cochlea, immunogold labeling reveals cadherin 23 is associated with kinocilial links and transient lateral links located between and within stereociliary rows. At this stage, the cadherin 23 ectodomain epitope remains on the hair bundle following BAPTA or La 3+ treatment, but is lost following exposure to the protease subtilisin. In contrast, mechano-electrical transduction is abolished by BAPTA but unaffected by subtilisin. These results suggest cadherin 23 is associated with transient lateral links that have properties distinct from those of the tip-link.

Spatiotemporal pattern and isoforms of cadherin 23 in wild type and waltzer mice during inner ear hair cell development

Mutant alleles of the gene encoding cadherin 23 are associated with Usher syndrome type 1 (USH1D), isolated deafness (DFNB12) in humans, and deafness and circling behavior in waltzer ( v) mice. Stereocilia of waltzer mice are disorganized and the kinocilia misplaced, indicating the importance of cadherin 23 for hair bundle development. Cadherin 23 was localized to developing stereocilia and proposed as a component of the tip link. We show that, during development of the inner ear, cadherin 23 is initially detected in centrosomes at E14.5, then along the length of emerging stereocilia, and later becomes concentrated at and subsequently disappears from the tops of stereocilia. In mature vestibular hair bundles, cadherin 23 is present along the kinocilium and in the region of stereocilia–kinocilium bonds, a pattern conserved in mammals, chicks, and frogs. Cadherin 23 is also present in Reissner's membrane (RM) throughout development. In homozygous v 6J mice, a reported null allele, cadherin 23 was absent from stereocilia, but present in kinocilia, RM, and centrosomes. We reconciled these results by identifying two novel isoforms of Cdh23 unaffected in sequence and expression by the v 6J allele. Our results suggest that Cdh23 participation in stereocilia links may be restricted to developing hair bundles.

Tonic mechanosensitivity of outer hair cells after loss of tip links

Tip links – the extracellular connectors between the distal ends of adjacent stereocilia – are essential for the fast mechanical gating of hair-cell transducer channels. Transduction in the absence of tip links was investigated for outer hair cells of the adult guinea-pig cochlea by patch-clamp recordings of the whole-cell current during mechanical stimulation of the hair bundle. Loss of tip links induced by application of BAPTA led to permanently opened transducer channels, as evidenced by a constant inward current, loss of response to sinusoidal mechanical deflection of the hair bundle and block by the open-channel blocker dihydrostreptomycin (100 μM). Step deflection of the hair bundle (200–500 nm) in the inhibitory direction exponentially reduced this current to a constant value with time constant, τon, of the order of seconds. The current returned exponentially to the pre-stimulus level with time-constant, τoff, also of the order of seconds. τon was dependent on the inter-stimulus interval, Δt, such that reducing this interval below about 40 s resulted in an exponentially faster response. τoff was independent of Δt. Application of the calcium ionophore, ionomycin (10 μM), showed that τon became independent of Δt after saturating elevation of the intracellular Ca2+ concentration. Flash-photolytic release of intracellular caged calcium (25-μM NP-EGTA/AM) showed that τon is dependent on intracellular Ca2+ concentration. These experiments imply an intracellular, calcium-dependent gating mechanism for hair-cell transducer channels.

Is there a close relationship between changes in amplitudes of distortion product otoacoustic emissions and hair cell damage after exposure to realistic industrial noise in guinea pigs?

In long-term experiments in awake guinea pigs (n = 12), distortion product otoacoustic emissions (DPOAEs) at various frequencies were measured repeatedly over 6-8 months. About 9 weeks after the first measurement, the animals were exposed to industrial noise (car industry, maximal intensity about 110 dB SPL) for 2 h. The amplitudes of DPOAE were measured prior to noise exposure and 10 min, 70 min, 1 day and 2 days after the noise exposure and then once every week. Three to four months after noise exposure, the animals were killed, and the cochleae were prepared for scanning electron microscopy. The row of inner hair cells (IHCs) was complete in all animals, while the rows of outer hair cells (OHCs) showed a considerable hair cell loss in some of the animals without a correlation to the change in amplitudes of DPOAE. However, a closer relationship between the decline of amplitudes of DPOAE and the number of missing and changed OHCs (fused stereocilia bundles, missing tip links) could be established. The number of lost OHC does not reflect the decline in DPOAE in all cases. This discrepancy must be considered when the degree of hearing loss needs to be established from changed DPOAE.

The Structure of Tip Links and Kinocilial Links in Avian Sensory Hair Bundles

Recent studies have indicated that the tip links and kinocilial links of sensory hair bundles in the inner ear have similar properties and share a common epitope, and that cadherin 23 may also be a component of each link type. Transmission electron microscopy was therefore used to study and compare the fine structure of the tip links and kinocilial links in avian sensory hair bundles. Tannic acid treatment revealed a thin strand, 150–200 nm long and 8–11 nm thick, present in both link types. Fourier analysis of link images showed that the strand of both link types is formed from two filaments coiled in a helix-like arrangement with an axial period of 20–25 nm, with each filament composed of globular structures that are ∼4 nm in diameter. Differences in the radius and period of the helix-like structure may underlie the observed variation in the length of tip and kinocilial links. The similar helix-like structure of the tip links and kinocilial links is in accord with the presence of a common cell-surface antigen (TLA antigen) and similarities in the physical and chemical properties of the two link types. The spacing of the globular structures comprising each filament of the two link types is similar to the 4.3 nm center-to-center spacing reported for the globular cadherin repeat, and is consistent with the suggestion that cadherin 23 is the tip link.

The developmental genetics of auditory hair cells.

Loss of auditory hair cells (AHCs) is a major cause of human deafness. Considerable effort has been devoted to unraveling how these mechanotransducers of sound are specified, with a view to correcting hearing loss by gene or stem cell therapies. Recent work on signaling cascades, particularly lateral inhibition and planar cell polarity, has begun to tie together some of the known pathways. Mutations in mice and humans that cause hearing and/or balance disorders are also shedding light on how AHCs are specified and, maintained and handle ion flux. Studies on some of these genes are beginning to provide insights into the more complex genetics of later onset forms of hearing loss. Progress has also been made in solving some long-term goals of auditory biology. Cadherin23 has been identified as a component of AHC stereocilia tip links, and progress has been made towards identifying the elusive AHC mechanoreceptor channel. Preliminary steps have also been taken towards inner-ear gene therapy, and in the engineering of embryonic stem cells for eventual cell therapies. Mammals cannot regenerate AHCs, but birds and other lower vertebrates can. Genomic tools have now been brought to bear on this problem with the aim of deciphering the molecular basis of this regenerative capability. The combination of new genomic tools and the many mouse and chicken embryological and genetic resources should increasingly provide new insights into how AHCs are programed and maintained.

Computational models of hair cell bundle mechanics: III. 3-D utricular bundles

Six utricular hair bundles from a red-eared turtle are modeled using 3-D finite element analysis. The mechanical model includes shear deformable stereocilia, realignment of all forces during force load increments, and tip and lateral link inter-stereocilia connections. Results show that there are two distinct bundle types that can be separated by mechanical bundle stiffness. The more compliant group has fewer total stereocilia and short stereocilia relative to kinocilium height; these cells are located in the medial and lateral extrastriola. The stiff group are located in the striola. They have more stereocilia and long stereocilia relative to kinocilia heights. Tip link tensions show parallel behavior in peripheral columns of the bundle and serial behavior in central columns when the tip link modulus is near or above that of collagen (1 × 10 9 N/m 2). This analysis shows that lumped parameter models of single stereocilia columns can show some aspects of bundle mechanics; however, a distributed, 3-D model is needed to explore overall bundle behavior.