Molecular mechanisms underlying function of hair bundle: study on genetic deafness in mouse models

Although the basic principles for the function of peripheral auditory system have been known for many years, the molecular mechanisms which affect deafness are not clear. In recent years, the study of hereditary deafness associated mouse models has revealed the molecular basis which is related with the formation and function of the hair bundle and the mechanosensory organelle of hair cell. This review focused on the role of protein network, which is formed by the proteins encoded by the Usher syndrome type 1 genes, in hair-bundle development and mechanotransducer channel gating. And the review also showed how the stereocilia rootlets contribute to the hair bundle's mechanical properties and how the hair bundle produces suppressive masking. Finally, the review revealed multiple roles of the tectorial membrane and extracellular matrix in the hair bundles stimulating in the cochlea.     

Stereocilia membrane deformation: implications for the gating spring and mechanotransduction channel

In hair cells, although mechanotransduction channels have been localized to tips of shorter stereocilia of the mechanically sensitive hair bundle, little is known about how force is transmitted to the channel. Here, we use a biophysical model of the membrane-channel complex to analyze the nature of the gating spring compliance and channel arrangement. We use a triangulated surface model and Monte Carlo simulation to compute the deformation of the membrane under the action of tip link force. We show that depending on the gating spring stiffness, the compliant component of the gating spring arises from either the membrane alone or a combination of the membrane and a tether that connects the channel to the actin cytoskeleton. If a bundle is characterized by relatively soft gating springs, such as those of the bullfrog sacculus, the need for membrane reinforcement by channel tethering then depends on membrane parameters. With stiffer gating springs, such as those from rat outer hair cells, the channel must be tethered for all biophysically realistic parameters of the membrane. We compute the membrane forces (resultants), which depend on membrane tension, bending modulus, and curvature, and show that they can determine the fate of the channel.     

Theoretical conditions for high-frequency hair bundle oscillations in auditory hair cells

Substantial evidence exists for spontaneous oscillations of hair cell stereociliary bundles in the lower vertebrate inner ear. Since the oscillations are larger than expected from Brownian motion, they must result from an active process in the stereociliary bundle suggested to underlie amplification of the sensory input as well as spontaneous otoacoustic emissions. However, their low frequency (<100 Hz) makes them unsuitable for amplification in birds and mammals that hear up to 5 kHz or higher. To examine the possibility of high-frequency oscillations, we used a finite-element model of the outer hair cell bundle incorporating previously measured mechanical parameters. Bundle motion was assumed to activate mechanotransducer channels according to the gating spring hypothesis, and the channels were regulated adaptively by Ca²⁺ binding. The model generated oscillations of freestanding bundles at 4 kHz whose sharpness of tuning depended on the mechanotransducer channel number and location, and the Ca²⁺ concentration. Entrainment of the oscillations by external stimuli was used to demonstrate nonlinear amplification. The oscillation frequency depended on channel parameters and was increased to 23 kHz principally by accelerating Ca²⁺ binding kinetics. Spontaneous oscillations persisted, becoming very narrow-band, when the hair bundle was loaded with a tectorial membrane mass.     

A virtual hair cell, II: evaluation of mechanoelectric transduction parameters

The virtual hair cell we have proposed utilizes a set of parameters related to its mechanoelectric transduction. In this work, we observed the effect of such channel gating parameters as the gating threshold, critical tension, resting tension, and Ca(2+) concentration. The gating threshold is the difference between the resting and channel opening tension exerted by the tip link assembly on the channel. The critical tension is the tension in the tip link assembly over which the channel cannot close despite Ca(2+) binding. Our results show that 1), the gating threshold dominated the initial sensitivity of the hair cell; 2), the critical tension minimally affects the peak response, (I), but considerably affects the time course of response, I(t), and the force-displacement, F-X, relationship; and 3), higher intracellular [Ca(2+)] resulted in a smaller fast adaptation time constant. Based on the simulation results we suggest a role of the resting tension: to help overcome the viscous drag of the hair bundle during the oscillatory movement of the bundle. Also we observed the three-dimensional bundle effect on the hair cell response by varying the number of cilia forced. These varying forcing conditions affected the hair cell response.     

A mechanical model of the gating spring mechanism of stereocilia

The stereocilium is the basic sensory unit of nature's mechanotransducers, which include the cochlear and vestibular organs. In noisy environments, stereocilia display high sensitivity to miniscule stimuli, effectively dealing with a situation that is a design challenge in micro systems. The gating spring hypothesis suggests that the mechanical stiffness of stereocilia bundle is softened by tip-link gating in combination with active bundle movement, contributing to the nonlinear amplification of miniscule stimuli. To demonstrate that the amplification is induced mechanically by the gating as hypothesized, we developed a biomimetic model of stereocilia and fabricated the model at the macro scale. The model consists of an inverted pendulum array with bistable buckled springs at its tips, which represent the mechanically gated ion channel. Model simulations showed that at the moment of gating, instantaneous stiffness softening generates an increase in response magnitude, which then sequentially occurs as the number of gating increases. This amplification mechanism appeared to be robust to the change of model parameters. Experimental data from the fabricated macro model also showed a significant increase in the open probability and pendulum deflection at the region having a smaller input magnitude. The results demonstrate that the nonlinear amplification of miniscule stimuli is mechanically produced by stiffness softening from channel gating.     

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 5,300 pN/microm, and the combined stiffness of shaft links binding two adjacent stereocilia of 750 approximately 2,250 pN/microm. 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.     

Friction from Transduction Channels’ Gating Affects Spontaneous Hair-Bundle Oscillations

Hair cells of the inner ear can power spontaneous oscillations of their mechanosensory hair bundle, resulting in amplification of weak inputs near the characteristic frequency of oscillation. Recently, dynamic force measurements have revealed that delayed gating of the mechanosensitive ion channels responsible for mechanoelectrical transduction produces a friction force on the hair bundle. The significance of this intrinsic source of dissipation for the dynamical process underlying active hair-bundle motility has remained elusive. The aim of this work is to determine the role of friction in spontaneous hair-bundle oscillations. To this end, we characterized key oscillation properties over a large ensemble of individual hair cells and measured how viscosity of the endolymph that bathes the hair bundles affects these properties. We found that hair-bundle movements were too slow to be impeded by viscous drag only. Moreover, the oscillation frequency was only marginally affected by increasing endolymph viscosity by up to 30-fold. Stochastic simulations could capture the observed behaviors by adding a contribution to friction that was 3?8-fold larger than viscous drag. The extra friction could be attributed to delayed changes in tip-link tension as the result of the finite activation kinetics of the transduction channels. We exploited our analysis of hair-bundle dynamics to infer the channel activation time, which was ～1 ms. This timescale was two orders-of-magnitude shorter than the oscillation period. However, because the channel activation time was significantly longer than the timescale of mechanical relaxation of the hair bundle, channel kinetics affected hair-bundle dynamics. Our results suggest that friction from channel gating affects the waveform of oscillation and that the channel activation time can tune the characteristic frequency of the hair cell. We conclude that the kinetics of transduction channels’ gating plays a fundamental role in the dynamic process that shapes spontaneous hair-bundle oscillations.     

Ion channels for the mechano-electrical transduction and efferent synapse of the hair cell

Auditory and vestibular information is applied to the hair cell hair bundle as mechanical energy, and is transduced into electrical energy by gating ion channels. The m-e.t. channel has a unitary conductance of 50 pS and a broad selectivity to monovalent cations and to divalent cations. Ca ions are the most permeable through the channel. The angular displacement of the hair bundle is the primary gating factor. Circumstantial evidence indicates the possibility of the direct gating of channels by the membrane deformation itself. The transduction potential activates voltage gated Ca channel and leads to the release of neurotransmitters which activate afferent neurones. Cholinergic muscarinic receptors likely mediate the inhibitory efferent innervation to the hair cell.     

Compliance of the hair bundle associated with gating of mechanoelectrical transduction channels in the bullfrog's saccular hair cell

Mechanical stimuli are thought to open the transduction channels of a hair cell by tensing elastic components, the gating springs, that pull directly on the channels. To test this model, we measured the stiffness of hair bundles during mechanical stimulation. A bundle's compliance increased by about 40% at the position where half of the channels opened. This we attribute to conformational changes of transduction channels as they open and close. The magnitude and displacement dependence of the gating compliance provide quantitative information about the molecular basis of mechanoelectrical transduction: the force required to open each channel, the number of transduction channels per hair cell, the stiffness of a gating spring, and the swing of a channel's gate as it opens.     

Development of the hair bundle and mechanotransduction

This review focuses on the cellular and molecular mechanisms underlying the development of the sensory hair bundle, an apical specialisation of the hair cell that is essential for mechanotransduction. The structure, function and development of the hair bundle is described, with an emphasis on the properties and possible roles played by the different link types that interconnect the individual elements of the hair bundle - the multiple stereocilia and the single kinocilium. Studies of mouse and zebrafish mutants have revealed that several classes of molecule are required for the genesis and maintenance of hair-bundle structure. These include cell surface molecules that are associated with the different hair-bundle links, along with myosin motors, scaffolding proteins and an actin cross-linker. Finally we consider how differences in the form and shape of hair bundles within and between different sensory organs are generated.     

A virtual hair cell, I: addition of gating spring theory into a 3-D bundle mechanical model

We have developed a virtual hair cell that simulates hair cell mechanoelectrical transduction in the turtle utricle. This study combines a full three-dimensional hair bundle mechanical model with a gating spring theory. Previous mathematical models represent the hair bundle with a single degree of freedom system which, we have argued, cannot fully explain hair bundle mechanics. In our computer model, the tip link tension and fast adaptation modulator kinetics determine the opening and closing of each channel independently. We observed the response of individual transduction channels with our presented model. The simulated results showed three features of hair cells in vitro. First, a transient rebound of the bundle tip appeared when fast adaptation dominated the dynamics. Second, the dynamic stiffness of the bundle was minimized when the response-displacement (I-X) curve was steepest. Third, the hair cell showed "polarity", i.e., activation decreased from a peak to zero as the forcing direction rotated from the excitatory to the inhibitory direction.     

Gating of Two Mechanoelectrical Transducer Channels Associated with a Single Tip Link

Although gating of mechanoelectrical transducer (MET) channels has been successfully described by assuming that one channel is associated with a tip link in the hair bundle, recent reports indicate that a single tip link is associated with more than one channel. To address the consistency of the model with the observations, gating of MET channels is described here by assuming that each tip link is associated with two identical MET channels, which are connected either in series or in parallel. We found that series connection does not lead to a single minimum of stiffness with respect to hair bundle displacement unless the minimum is above a certain positive value. Thus, negative stiffness must appear in pairs in the displacement axis. In contrast, parallel connection of the two channels predicts gating compliance similar to that predicted by the one-channel-per-tip-link model of channel gating, within the physiological range of parameters. Parallel connection of MET channels is, therefore, a reasonable assumption to explain most experimental observations. However, the compatibility with series connection cannot be ruled out for experimental data on turtle hair cells.     

Unconventional Mechanics of Lipid Membranes: A Potential Role for Mechanotransduction of Hair Cell Stereocilia

A force-conveying role of the lipid membrane across various mechanoreceptors is now an accepted hypothesis. However, such a mechanism is still not fully understood for mechanotransduction in the hair bundle of auditory sensory hair cells. A major goal of this theoretical assessment was to investigate the role of the lipid membrane in auditory mechanotransduction, especially in generating nonlinear bundle force versus displacement measurements, one of the main features of auditory mechanotransduction. To this end, a hair bundle model that generates lipid membrane tented deformation in the stereocilia was developed. A computational analysis of the model not only reproduced nonlinear bundle force measurements but also generated membrane energy that is potentially sufficient to activate the mechanosensitive ion channel of the hair cell. In addition, the model provides biophysical insight into 1) the likelihood that the channel must be linked in some way to the tip link; 2) how the interplay of the bending and stretching of the lipid bilayer may be responsible for the nonlinear force versus displacement response; 3) how measurements of negative stiffness may be a function of the rotational stiffness of the rootlets; and 4) how the standing tension of the tip link is required to interpret migration of the nonlinear force versus displacement and activation curves. These are all features of hair cell mechanotransduction, but the underlying biophysical mechanism has proved elusive for the last three decades.     

Nonlinear mechanical responses of mouse cochlear hair bundles.

The stiffness of sensory hair bundles of both inner (IHC) and outer (OHC) hair cells was measured with calibrated silica fibres in mouse cochlear cultures to test the hypothesis that the mechanical properties of the hair bundle reflect processes underlying mechanotransduction. For OHCs, the displacement of the hair bundle relaxed with time constants of 6 ms for displacements which open transducer channels and 4 ms for displacements which close the channels. The corresponding values of the time constants for IHCs were 10 ms and 8 ms, respectively. A displacement-dependent change in the stiffness of the hair bundle was not observed when the bundle was displaced orthogonally to the direction of excitation. The stiffness of the hair bundle as a function of nanometre displacements from the resting position was remarkably nonlinear. The stiffness declined to a minimum from the resting stiffness by about 12% for OHCs and 20% for IHCs when the hair bundle was displaced by about 20 nm in the excitatory direction, and it increased by a similar amount when the bundle was displaced by 20 nm in the inhibitory direction. The displacement at which the stiffness reached a minimum was within the most sensitive region of the hair-cell transducer function (receptor potential as a function of hair-bundle displacement), and the displacement at which the stiffness reached a maximum was at the point of saturation of the transducer function in the inhibitory direction. The nonlinear displacement-dependent compliance change is reversibly abolished, and the time constant of relaxation of the bundle for excitatory displacements is reversibly reduced, when mechanotransduction is blocked by the addition of either neomycin sulphate or cobalt chloride to the solution bathing the hair cells. The displacement-dependent compliance change was not apparently reduced when the receptor potential was attenuated through the substitution of sodium in the bathing solution with a less permeant cation, tetraethylammonium. These findings suggest that the nonlinear mechanical properties of the hair bundle are associated with aspects of the hair-cell mechanotransducer process. The mechanical properties of the hair bundle are discussed in relation to the 'gating-spring' hypothesis of hair-cell transduction.     

Fundamental role of microvilli in the main functions of differentiated cells: Outline of an universal regulating and signaling system at the cell periphery

Until now, the general importance of microvilli present on the surface of almost all differentiated cells has been strongly underestimated and essential functions of these abundant surface organelles remained unrecognized. Commonly, the role of microvilli has been reduced to their putative function of cell‐surface enlargement. In spite of a large body of detailed knowledge about the specific functions of microvilli in sensory receptor cells for sound, light, and odor perception, their functional importance for regulation of basic cell functions remained obscure. Here, a number of microvillar mechanisms involved in fundamental cell functions are discussed. Two structural features enable the extensive functional competence of microvilli: First, the exclusive location of almost all functional important membrane proteins on microvilli of differentiated cells and second, the function of the F‐actin‐based cytoskeletal core of microvilli as a structural diffusion barrier modulating the flow of low molecular substrates and ions into and out of the cell. The specific localization on microvilli of important functional membrane proteins such as glucose transporters, ion channels, ion pumps, and ion exchangers indicate the importance and diversity of microvillar functions. In this review, the microvillar mechanisms of audioreceptor, photoreceptor, and olfactory/taste receptor cells are discussed as highly specialized adaptations of a general type of microvillar mechanisms involved in regulation of important basic cell functions such as glucose transport/energy metabolism, ion channel regulation, generation and modulation of the membrane potential, volume regulation, and Ca signaling. Even the constitutive cellular defence against cytotoxic compounds, also called “multidrug resistance (MDR),” is discussed as a microvillar mechanism. A comprehensive examination of the specific properties of “cable‐like” ion conduction along the microvillar core structure of F‐actin allows the proposal that microvilli are specifically designed for using ionic currents as cellular signals. In view of the multifaceted gating and signaling properties of TRP channels, the possible role of microvilli as a universal gating device for TRP channel regulation is discussed. Combined with the role of the microvillar core bundle of actin filaments as high‐affinity Ca store, microvilli may turn out as highly specialized Ca signaling organelle involved in store‐operated Ca entry (SOCE) and initiation of nonlinear Ca signals such as waves and oscillations. J. Cell. Physiol. 226: 896–927, 2011. © 2010 Wiley‐Liss, Inc.     

Mechano-electrical transducer currents in hair cells of the cultured neonatal mouse cochlea.

The first step towards the generation of the receptor potential in hair cells is the gating of the transducer channels and subsequent flow of transducer current, induced by deflection of the stereocilia. We describe properties of the transducer current in outer hair cells of neonatal mice. Less extensive observations on inner hair cells suggest that their transducer currents have similar characteristics. The hair bundles were stimulated by force from a fluid jet. The transducer currents in outer hair cells are the largest found so far in any hair cell, with a chord conductance of up to 9.2 nS at -84 mV. The transfer function suggests that the channel has at least two closed states and one open state. The permeabilities for sodium, potassium and caesium are similar, consistent with the channel being a fairly non-selective cation channel. At negative potentials the currents adapt in most cells, although never as completely as in hair cells of lower vertebrates. If the unit conductance of the transducer channel is similar to that of the turtle's auditory hair cells (100 pS), then there are about 90 channels per hair bundle, or one channel between every pair of adjacent stereocilia in neighbouring rows.     

In search of the hair-cell gating spring elastic properties of ankyrin and cadherin repeats

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.     

Effectiveness of Hair Bundle Motility as the Cochlear Amplifier

The effectiveness of hair bundle motility in mammalian and avian ears is studied by examining energy balance for a small sinusoidal displacement of the hair bundle. The condition that the energy generated by a hair bundle must be greater than energy loss due to the shear in the subtectorial gap per hair bundle leads to a limiting frequency that can be supported by hair-bundle motility. Limiting frequencies are obtained for two motile mechanisms for fast adaptation, the channel re-closure model and a model that assumes that fast adaptation is an interplay between gating of the channel and the myosin motor. The limiting frequency obtained for each of these models is an increasing function of a factor that is determined by the morphology of hair bundles and the cochlea. Primarily due to the higher density of hair cells in the avian inner ear, this factor is ∼10-fold greater for the avian ear than the mammalian ear, which has much higher auditory frequency limit. This result is consistent with a much greater significance of hair bundle motility in the avian ear than that in the mammalian ear.     

The actions of calcium on hair bundle mechanics in mammalian cochlear hair cells

Sound stimuli excite cochlear hair cells by vibration of each hair bundle, which opens mechanotransducer (MT) channels. We have measured hair-bundle mechanics in isolated rat cochleas by stimulation with flexible glass fibers and simultaneous recording of the MT current. Both inner and outer hair-cell bundles exhibited force-displacement relationships with a nonlinearity that reflects a time-dependent reduction in stiffness. The nonlinearity was abolished, and hair-bundle stiffness increased, by maneuvers that diminished calcium influx through the MT channels: lowering extracellular calcium, blocking the MT current with dihydrostreptomycin, or depolarizing to positive potentials. To simulate the effects of Ca²⁺, we constructed a finite-element model of the outer hair cell bundle that incorporates the gating-spring hypothesis for MT channel activation. Four calcium ions were assumed to bind to the MT channel, making it harder to open, and, in addition, Ca²⁺ was posited to cause either a channel release or a decrease in the gating-spring stiffness. Both mechanisms produced Ca²⁺ effects on adaptation and bundle mechanics comparable to those measured experimentally. We suggest that fast adaptation and force generation by the hair bundle may stem from the action of Ca²⁺ on the channel complex and do not necessarily require the direct involvement of a myosin motor. The significance of these results for cochlear transduction and amplification are discussed.