HCN Channels Are Not Required for Mechanotransduction in Sensory Hair Cells of the Mouse Inner Ear

The molecular composition of the hair cell transduction channel has not been identified. Here we explore the novel hypothesis that hair cell transduction channels include HCN subunits. The HCN family of ion channels includes four members, HCN1-4. They were orginally identified as the molecular correlates of the hyperpolarization-activated, cyclic nucleotide gated ion channels that carry currents known as I_f, I_Q or I_h. However, based on recent evidence it has been suggested that HCN subunits may also be components of the elusive hair cell transduction channel. To investigate this hypothesis we examined expression of mRNA that encodes HCN1-4 in sensory epithelia of the mouse inner ear, immunolocalization of HCN subunits 1, 2 and 4, uptake of the transduction channel permeable dye, FM1-43 and electrophysiological measurement of mechanotransduction current. Dye uptake and transduction current were assayed in cochlear and vestibular hair cells of wildtype mice exposed to HCN channel blockers or a dominant-negative form of HCN2 that contained a pore mutation and in mutant mice that lacked HCN1, HCN2 or both. We found robust expression of HCNs 1, 2 and 4 but little evidence that localized HCN subunits in hair bundles, the site of mechanotransduction. Although high concentrations of the HCN antagonist, ZD7288, blocked 50–70% of the transduction current, we found no reduction of transduction current in either cochlear or vestibular hair cells of HCN1- or HCN2- deficient mice relative to wild-type mice. Furthermore, mice that lacked both HCN1 and HCN2 also had normal transduction currents. Lastly, we found that mice exposed to the dominant-negative mutant form of HCN2 had normal transduction currents as well. Taken together, the evidence suggests that HCN subunits are not required for mechanotransduction in hair cells of the mouse inner ear.     

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.     

Vertebrate and invertebrate TRPV-like mechanoreceptors

Our senses of touch, hearing, and balance are mediated by mechanosensitive ion channels. In vertebrates, little is known about the molecular composition of these mechanoreceptors, an example of which is the transduction channel of the inner ear's receptor cells, hair cells. Members of the TRP family of ion channels are considered candidates for the vertebrate hair cell's mechanosensitive transduction channel and here we review the evidence for this candidacy. We start by examining the results of genetic screens in invertebrates that identified members of the TRP gene family as core components of mechanoreceptors. In particular, we discuss the Caenorhabditis elegans OSM-9 channel, an invertebrate TRPV channel, and the Drosophila melanogaster TRP channel NOMPC. We then evaluate basic features of TRPV4, a vertebrate member of the TRPV subfamily, which is gated by a variety of physical and chemical stimuli including temperature, osmotic pressure, and ligands. Finally, we compare the characteristics of all discussed mechanoreceptive TRP channels with the biophysical characteristics of hair cell mechanotransduction, speculating about the possible make-up of the elusive inner ear mechanoreceptor.     

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.     

Fast adaptation in vestibular hair cells requires myosin-1c activity

In sensory hair cells of the inner ear, mechanical amplification of small stimuli requires fast adaptation, the rapid closing of mechanically activated transduction channels. In frog and mouse vestibular hair cells, we found that the rate of fast adaptation depends on both channel opening and stimulus size and that it is modeled well as a release of a mechanical element in series with the transduction apparatus. To determine whether myosin-1c molecules of the adaptation motor are responsible for the release, we introduced the Y61G mutation into the Myo1c locus and generated mice homozygous for this sensitized allele. Measuring transduction and adaptation in the presence of NMB-ADP, an allele-specific inhibitor, we found that the inhibitor not only blocked slow adaptation, as demonstrated previously in transgenic mice, but also inhibited fast adaptation. These results suggest that mechanical activity of myosin-1c is required for fast adaptation in vestibular hair cells.     

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.     

Localization of the hair cell's transduction channels at the hair bundle's top by iontophoretic application of a channel blocker.

In order to understand how the hair cell's mechanoelectrical transduction channels are gated during mechanical stimulation, it is essential to determine their location with respect to the hair bundle's constituent stereocilia. We localized the transduction channels by focally blocking receptor currents with iontophoretically ejected gentamicin, an aminoglycoside antibiotic that acts as a reversible channel blocker. The drug was most effective when directed at the top of a hair bundle, whereas application at the bundle's bottom or at the cuticular plate had little or no effect. Computer simulations of blocking agreed with experimental data only when the transduction channels were hypothesized to occur near the bundle's top. These results confirm that the hair cell's transduction channels are located near the stereociliary tips.     

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.     

Hypotonic stimuli enhance proton-gated currents of acid-sensing ion channel-1b

Acid-sensing ion channels (ASICs) are strong candidates for mammalian mechanoreceptors. We investigated whether mouse acid-sensing ion channel-1b (ASIC1b) is sensitive to mechanical stimuli using oocyte electrophysiology, because ASIC1b is located in the mechanosensory stereocilia of cochlear hair cells. Hypotonic stimuli that induced membrane stretch of oocytes evoked no significant current in ASIC1b-expressing oocytes at pH 7.5. However, acid (pH 4.0 or 5.0)-evoked currents in the oocytes were substantially enhanced by the hypotonicity, showing mechanosensitivity of ASIC1b and possible mechanogating of the channel in the presence of other components. Interestingly, the ASIC1b channel was permeable to K(+) (a principal charge carrier for cochlear sensory transduction) and the affinity of the channel for amiloride (IC(50) (inhibition constant)=approximately 48.3 microM) was quite similar to that described for the mouse hair cell mechanotransducer current. Taken together, these data raise the possibility that ASIC1b participates in cochlear mechanoelectrical transduction.     

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.     

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.     

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.     

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.     

Have we found the tip link,transduction channel,and gating spring of the hair cell?

Recent reports have offered candidates for key components of the apparatus used for mechanotransduction in hair cells. TRPA1 and cadherin 23 have been proposed to be the transduction channel and component of the tip link, respectively; moreover, ankyrin repeats in TRPA1 have been proposed to be the gating spring. Although these are excellent candidates for the three components, definitive experiments supporting each identification have yet to be performed.     

Amiloride causes changes in the mechanical properties of hair cell bundles in the fish lateral line similar to those induced by dihydrostreptomycin

Amiloride is a known blocker of the mechano-electrical transduction current in sensory hair cells. Measurements of cupular motion in the lateral line organ of fish now show that amiloride concurrently changes the micromechanical properties of the hair cell bundles. The effects of amiloride on the mechanics and receptor potentials of the hair cells resemble those previously observed for the aminoglycoside drug dihydrostreptomycin (DHSM) and are similarly antagonized by Ca²⁺. We hypothesize that amiloride and DHSM act on hair cells in two correlated ways which manifest themselves in both the electrical and mechanical properties of the transduction process. One action is the reduction of the transduction current with a concurrent increase of the hair bundle stiffness. The other action is a shift of the hair cell''s operating point on a current–displacement curve, with a concomitant shift along the associated hair bundle stiffness–displacement curve. The latter action has the opposite effect to that of the first and thus may lead, at relatively low blocker concentrations, to both an increase of transduction current and a decrease in hair bundle stiffness.     

Myosin VIIA defects, which underlie the Usher 1B syndrome in humans, lead to deafness in Drosophila

In vertebrates, auditory and vestibular transduction occurs on apical projections (stereocilia) of specialized cells (hair cells). Mutations in myosin VIIA (myoVIIA), an unconventional myosin, lead to deafness and balance anomalies in humans, mice, and zebrafish; individuals are deaf, and stereocilia are disorganized. The exact mechanism through which myoVIIA mutations result in these inner-ear anomalies is unknown. Proposed inner-ear functions for myoVIIA include anchoring transduction channels to the stereocilia membrane, trafficking stereocilia linking components, and anchoring hair cells by associating with adherens junctions. The Drosophila myoVIIA homolog is crinkled (ck). The Drosophila auditory organ, Johnston's organ (JO), is developmentally and functionally related to the vertebrate inner ear. Both derive from modified epithelial cells specified by atonal and spalt homolog expression, and both transduce acoustic mechanical energy (and references therein). Here, we show that loss of ck/myoVIIA function leads to complete deafness in Drosophila by disrupting the integrity of the scolopidia that transduce auditory signals. We demonstrate that ck/myoVIIA functions to organize the auditory organ, that it is functionally required in neuronal and support cells, that it is not required for TRPV channel localization, and that it is not essential for scolopidial-cell-junction integrity.     

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.     

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.     

Hair Cells – Beyond the Transducer

Overview This review considers the “tween twixt and twain” of hair cell physiology, specifically the signaling elements and membrane conductances which underpin forward and reverse transduction at the input stage of hair cell function and neurotransmitter release at the output stage. Other sections of this review series outline the advances which have been made in understanding the molecular physiology of mechanoelectrical transduction and outer hair cell electromotility. Here we outline the contributions of a considerable array of ion channels and receptor signaling pathways that define the biophysical status of the sensory hair cells, contributing to hair cell development and subsequently defining the operational condition of the hair cells across the broad dynamic range of physiological function.