Ca2+ changes the force sensitivity of the hair-cell transduction channel

The mechanically gated transduction channels of vertebrate hair cells tend to close in approximately 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 approximately 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.     

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.     

Adaptation in auditory hair cells

The narrow stimulus limits of hair cell transduction, equivalent to a total excursion of about 100nm at the tip of the hair bundle, demand tight regulation of the mechanical input to ensure that the mechanoelectrical transducer (MET) channels operate in their linear range. This control is provided by multiple components of Ca(2+)-dependent adaptation. A slow mechanism limits the mechanical stimulus through the action of one or more unconventional myosins. There is also a fast, sub-millisecond, Ca(2+) regulation of the MET channel, which can generate resonance and confer tuning on transduction. Changing the conductance or kinetics of the MET channels can vary their resonant frequency. The tuning information conveyed in transduction may combine with the somatic motility of outer hair cells to produce an active process that supplies amplification and augments frequency selectivity in the mammalian cochlea.     

Characterization of adaptation motors in saccular hair cells by fluctuation analysis

The mechanical sensitivity of hair cells, the sensory receptors of the vestibular and auditory systems, is maintained by adaptation, which resets the transducer to cancel the effects of static stimuli. Adaptation motors in hair cells can be experimentally activated by externally applying a transduction channel blocker to the hair bundle, causing the hair bundle to move in the negative direction. We studied the variance in the position of the hair bundle during these displacements and found that it increases as the bundle moves to its new position. Often the variance peaks, and then declines to a steady-state value. We describe both displacement and variance with a model in which a motor acting on the bundle takes approximately 3.6-nm steps whose frequency (approximately 22 s(-1)) declines with the motor's load.     

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.     

Two adaptation processes in auditory hair cells together can provide an active amplifier

The hair cells of the vertebrate inner ear convert mechanical stimuli to electrical signals. Two adaptation mechanisms are known to modify the ionic current flowing through the transduction channels of the hair bundles: a rapid process involves Ca(2+) ions binding to the channels; and a slower adaptation is associated with the movement of myosin motors. We present a mathematical model of the hair cell which demonstrates that the combination of these two mechanisms can produce "self-tuned critical oscillations", i.e., maintain the hair bundle at the threshold of an oscillatory instability. The characteristic frequency depends on the geometry of the bundle and on the Ca(2+) dynamics, but is independent of channel kinetics. Poised on the verge of vibrating, the hair bundle acts as an active amplifier. However, if the hair cell is sufficiently perturbed, other dynamical regimes can occur. These include slow relaxation oscillations which resemble the hair bundle motion observed in some experimental preparations.     

Eps8 regulates hair bundle length and functional maturation of mammalian auditory hair cells

Hair cells of the mammalian cochlea are specialized for the dynamic coding of sound stimuli. The transduction of sound waves into electrical signals depends upon mechanosensitive hair bundles that project from the cell's apical surface. Each stereocilium within a hair bundle is composed of uniformly polarized and tightly packed actin filaments. Several stereociliary proteins have been shown to be associated with hair bundle development and function and are known to cause deafness in mice and humans when mutated. The growth of the stereociliar actin core is dynamically regulated at the actin filament barbed ends in the stereociliary tip. We show that Eps8, a protein with actin binding, bundling, and barbed-end capping activities in other systems, is a novel component of the hair bundle. Eps8 is localized predominantly at the tip of the stereocilia and is essential for their normal elongation and function. Moreover, we have found that Eps8 knockout mice are profoundly deaf and that IHCs, but not OHCs, fail to mature into fully functional sensory receptors. We propose that Eps8 directly regulates stereocilia growth in hair cells and also plays a crucial role in the physiological maturation of mammalian cochlear IHCs. Together, our results indicate that Eps8 is critical in coordinating the development and functionality of mammalian auditory hair cells.     

Hair cells require phosphatidylinositol 4,5-bisphosphate for mechanical transduction and adaptation

After opening in response to mechanical stimuli, hair cell transduction channels adapt with fast and slow mechanisms that each depend on Ca(2+). We demonstrate here that transduction and adaptation require phosphatidylinositol 4,5-bisphosphate (PIP(2)) for normal kinetics. PIP(2) has a striking distribution in hair cells, being excluded from the basal region of hair bundles and apical surfaces of frog saccular hair cells. Localization of a phosphatidylinositol lipid phosphatase, Ptprq, to these PIP(2)-free domains suggests that Ptprq maintains low PIP(2) levels there. Depletion of PIP(2) by inhibition of phosphatidylinositol 4-kinase or sequestration by aminoglycosides reduces the rates of fast and slow adaptation. PIP(2) and other anionic phospholipids bind directly to the IQ domains of myosin-1c, the motor that mediates slow adaptation, permitting a strong interaction with membranes and likely regulating the motor's activity. PIP(2) depletion also causes a loss in transduction current. PIP(2) therefore plays an essential role in hair cell adaptation and transduction.     

Lateral mechanical coupling of stereocilia in cochlear hair bundles

For understanding the gating process of transduction channels in the inner ear it is essential to characterize and examine the functional properties of the ultrastructure of stereociliary bundles. There is strong evidence that transduction channels in hair cells are gated by directly pulling at the so-called tip links. In addition to these tip links a second class of filamentous structures was identified in the scanning and transmission electron microscope: the side-to-side links. These links laterally connect stereocilia of the same row of a hair bundle. This study concentrates on mechanical coupling of stereocilia of the tallest row connected by side-to-side links. Atomic Force microscopy (AFM) was used to investigate hair bundles of outer hair cells (OHCs) from postnatal rats (day 4). Although hair bundles of postnatal rats are still immature at day 4 and interconnecting cross-links do not show preferential direction yet, hair bundles of investigated OHCs already showed the characteristic V-shape of mature hair cells. In a first experiment, the stiffness of stereocilia was investigated scanning individual stereocilia with an AFM tip. The spring constant for the excitatory direction was 2.5 +/- 0.6 x 10(-3) N/m whereas a higher spring constant (3.1 +/- 1.5 x 10(-3) N/m) was observed in the inhibitory direction. In a second set of experiments, the force transmission between stereocilia of the tallest row was measured using AFM in combination with a thin glass fiber. This fiber locally displaced a stereocilium while the force laterally transmitted to the neighboring untouched taller stereocilia was measured by AFM. The results show a weak force interaction between tallest stereocilia of postnatal rats. The force exerted to an individual stereocilium declines to 36% at the nearest adjacent stereocilium of the same row not touched with the fiber. It is suggested that the amount of force transmitted from a taller stereocilium to an adjacent one of the same row depends on the orientation of links. Maximum force transmission is expected to appear along the axis of interconnecting side links. In our studies it is suggested that transmitted forces are small because connecting side links are oriented very close to an angle of 90 degrees with respect of the scan direction (excitatory-inhibitory direction).     

Tip-link integrity and mechanical transduction in vertebrate hair cells.

An attractive hypothesis for hair-cell transduction is that fine, filamentous "tip links" pull directly on mechanically sensitive ion channels located at the tips of the stereocilia. We tested the involvement of tip links in the transduction process by treating bundles with a BAPTA-buffered, low-Ca2+ saline (10(-9) M). BAPTA abolished the transduction current in a few hundred milliseconds. BAPTA treatment for a few seconds eliminated the tip links observed by either scanning or transmission electron microscopy. BAPTA also eliminated the voltage-dependent movement and caused a positive bundle displacement of 133 nm, in quantitative agreement with a model for regulation of tension. We conclude that tip links convey tension to the transduction channels of hair cells.     

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.     

Coupling active hair bundle mechanics, fast adaptation, and somatic motility in a cochlear model

One of the central questions in the biophysics of the mammalian cochlea is determining the contributions of the two active processes, prestin-based somatic motility and hair bundle (HB) motility, to cochlear amplification. HB force generation is linked to fast adaptation of the transduction current via a calcium-dependent process and somatic force generation is driven by the depolarization caused by the transduction current. In this article, we construct a global mechanical-electrical-acoustical mathematical model of the cochlea based on a three-dimensional fluid representation. The global cochlear model is coupled to linearizations of nonlinear somatic motility and HB activity as well as to the micromechanics of the passive structural and electrical elements of the cochlea. We find that the active HB force alone is not sufficient to power high frequency cochlear amplification. However, somatic motility can overcome resistor-capacitor filtering by the basolateral membrane and deliver sufficient mechanical energy for amplification at basal locations. The results suggest a new theory for high frequency active cochlear mechanics, in which fast adaptation controls the transduction channel sensitivity and thereby the magnitude of the energy delivered by somatic motility.     

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.     

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.     

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.     

Molecular Remodeling of Tip Links Underlies Mechanosensory Regeneration in Auditory Hair Cells

Sound detection by inner ear hair cells requires tip links that interconnect mechanosensory stereocilia and convey force to yet unidentified transduction channels. Current models postulate a static composition of the tip link, with protocadherin 15 (PCDH15) at the lower and cadherin 23 (CDH23) at the upper end of the link. In terminally differentiated mammalian auditory hair cells, tip links are subjected to sound-induced forces throughout an organism''s life. Although hair cells can regenerate disrupted tip links and restore hearing, the molecular details of this process are unknown. We developed a novel implementation of backscatter electron scanning microscopy to visualize simultaneously immuno-gold particles and stereocilia links, both of only a few nanometers in diameter. We show that functional, mechanotransduction-mediating tip links have at least two molecular compositions, containing either PCDH15/CDH23 or PCDH15/PCDH15. During regeneration, shorter tip links containing nearly equal amounts of PCDH15 at both ends appear first. Whole-cell patch-clamp recordings demonstrate that these transient PCDH15/PCDH15 links mediate mechanotransduction currents of normal amplitude but abnormal Ca²⁺-dependent decay (adaptation). The mature PCDH15/CDH23 tip link composition is re-established later, concomitant with complete recovery of adaptation. Thus, our findings provide a molecular mechanism for regeneration and maintenance of mechanosensory function in postmitotic auditory hair cells and could help identify elusive components of the mechanotransduction machinery.     

ATPase activity of myosin in hair bundles of the bullfrog''s sacculus.

Mechanoelectrical transduction by a hair cell displays adaptation, which is thought to occur as myosin-based molecular motors within the mechanically sensitive hair bundle adjust the tension transmitted to transduction channels. To assess the enzymatic capabilities of the myosin isozymes in hair bundles, we examined the actin-dependent ATPase activity of bundles isolated from the bullfrog's sacculus. Separation of 32P-labeled inorganic phosphate from unreacted [gamma-32P]ATP by thin-layer chromatography enabled us to measure the liberation of as little as 0.1 fmol phosphate. To distinguish the Mg(2+)-ATPase activity of myosin isozymes from that of other hair-bundle enzymes, we inhibited the interaction of hair-bundle myosin with actin and determined the reduction in ATPase activity. N-ethylmaleimide (NEM) decreased neither physiologically measured adaptation nor the nucleotide-hydrolytic activity of a 120-kDa protein thought to be myosin 1 beta. The NEM-insensitive, actin-activated ATPase activity of myosin increased from 1.0 fmol x s-1 in 1 mM EGTA to 2.3 fmol x s-1 in 10 microM Ca2+. This activity was largely inhibited by calmidazolium, but was unaffected by the addition of exogenous calmodulin. These results, which indicate that hair bundles contain enzymatically active, Ca(2+)-sensitive myosin molecules, are consistent with the role of Ca2+ in adaptation and with the hypothesis that myosin forms the hair cell's adaptation motor.     

A core cochlear phenotype in USH1 mouse mutants implicates fibrous links of the hair bundle in its cohesion, orientation and differential growth

The planar polarity and staircase-like pattern of the hair bundle are essential to the mechanoelectrical transduction function of inner ear sensory cells. Mutations in genes encoding myosin VIIa, harmonin, cadherin 23, protocadherin 15 or sans cause Usher syndrome type I (USH1, characterized by congenital deafness, vestibular dysfunction and retinitis pigmentosa leading to blindness) in humans and hair bundle disorganization in mice. Whether the USH1 proteins are involved in common hair bundle morphogenetic processes is unknown. Here, we show that mouse models for the five USH1 genetic forms share hair bundle morphological defects. Hair bundle fragmentation and misorientation (25-52 degrees mean kinociliary deviation, depending on the mutant) were detected as early as embryonic day 17. Abnormal differential elongation of stereocilia rows occurred in the first postnatal days. In the emerging hair bundles, myosin VIIa, the actin-binding submembrane protein harmonin-b, and the interstereocilia-kinocilium lateral link components cadherin 23 and protocadherin 15, all concentrated at stereocilia tips, in accordance with their known in vitro interactions. Soon after birth, harmonin-b switched from the tip of the stereocilia to the upper end of the tip link, which also comprises cadherin 23 and protocadherin 15. This positional change did not occur in mice deficient for cadherin 23 or protocadherin 15. We suggest that tension forces applied to the early lateral links and to the tip link, both of which can be anchored to actin filaments via harmonin-b, play a key role in hair bundle cohesion and proper orientation for the former, and in stereociliary elongation for the latter.     

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.     

Poster Sessions BP08: Cytoskeleton and Molecular Motors. Sticking out its neck: myosin‐1c binding to hair‐cell receptors requires its IQ domains and is regulated by calmodulin

As the sensory receptor cells of the inner ear, hair cells transduce mechanical stimuli into electrical signals. While the electrophysiological properties of hair cells are well understood, little is known about the molecular basis of mechanoelectrical transduction. In particular, the identities of the transduction channel and other components of the transduction machinery are unknown. Myosin‐1c, an unconventional myosin formerly called myosin‐1 beta, is one identified component of the hair‐cell transduction apparatus where it functions in the adaptation process. Because of its role in adaptation, myosin‐1c must interact, either directly or indirectly, with other components of the transduction machinery, including the channel itself. As a result, we sought to characterize myosin‐1c receptors in hair cells. Using recombinant fragments of myosin‐1c with its associated calmodulin light chains, we examined binding of these complexes to myosin‐1c receptors in the hair bundle, the mechanosensitive organelle housing the transduction apparatus. Astonishingly, binding to hair‐cell receptors was not dependent upon the myosin's tail sequences, the domain of the protein long proposed to bind to intracellular cargo. Instead, binding to intracellular receptors depends upon the neck region of myosin‐1c which contains four calmodulin‐binding IQ domains. Calmodulin blocks myosin‐1c interaction with its receptors by binding to the myosin and obscuring the myosin‐1c/receptor interaction domain. The calcium‐sensitive binding of calmodulin to myosin‐1c may therefore regulate myosin‐1c interactions with other components of the transduction apparatus.