The frequency selectivity of mammalian hearing depends on not only the passive mechanics of the basilar membrane but also an active amplification of the mechanical stimulus by the cochlear hair cells. The common view is that amplification stems from the somatic motility of the outer hair cells (OHCs), changes in their length impelled by voltage-dependent transitions in the membrane protein prestin. Whether this voltage-controlled mechanism, whose frequency range may be limited by the membrane time constant, has the band width to cover the entire auditory range of mammals is uncertain. However, there is ample evidence for an alternative mode of force generation by hair cells of non-mammals, such as frogs and turtles, which probably lack prestin. The latter process involves active motion of the hair bundle underpinned by conformational changes in the mechanotransducer (MT) channels and activation of one or more isoforms of myosin. This review summarizes evidence for active hair bundle motion and its connection to MT channel adaptation. Key factors for the hair bundle motor to play a role in the mammalian cochlea include the size and speed of force production.

There is current debate about the origin of mechanical amplification whereby outer hair cells generate force to augment the sensitivity and frequency selectivity of the mammalian cochlea. To distinguish contributions to force production from the mechanotransducer (MET) channels and somatic motility, we have measured hair bundle motion during depolarization of individual outer hair cells in isolated rat cochleas. Depolarization evoked rapid positive bundle deflections that were reduced by perfusion with the MET channel blocker dihydrostreptomycin, with no effect on the nonlinear capacitance that is a manifestation of prestin-driven somatic motility. However, the movements were also diminished by Na salicylate and depended on the intracellular anion, properties implying involvement of the prestin motor. Furthermore, depolarization of one outer hair cell caused motion of neighboring hair bundles, indicating overall motion of the reticular lamina. Depolarization of solitary outer hair cells caused cell-length changes whose voltage-activation range depended on the intracellular anion but were insensitive to dihydrostreptomycin. These results imply that both the MET channels and the somatic motor participate in hair bundle motion evoked by depolarization. It is conceivable that the two processes can interact, a signal from the MET channels being capable of modulating the activity of the prestin motor.

To understand and model the contribution of outer hair cells to cochlear function, a full knowledge of both forward and reverse transduction is needed. To determine the limitations imposed by forward transduction, we have characterized mechanotransducer (MET) currents and hair bundle mechanics of outer hair cells in isolated coils of neonatal rats. In response to step deflections of the hair bundle, MET currents activated and then rapidly adapted with a sub-millisecond time constant that depended on extracellular calcium and cochlear location. The adaptation time constant imposed a first order high-pass filter at a corner frequency that, when corrected to in vivo conditions, was similar to the cell's characteristic frequency. Hair bundle mechanics determined by stimulation with a flexible fiber, exhibited a pronounced non-linearity that developed with the same time course as fast adaptation. We propose that the MET channels will high-pass filter the input signal and provide positive mechanical feedback to augment motion of the tectorial membrane. The time course of the feedback varies with cochlear location.

It is generally accepted that the acute sensitivity and frequency discrimination of mammalian hearing requires active mechanical amplification of the sound stimulus within the cochlea(1). The prevailing hypothesis is that this amplification stems from somatic electromotility of the outer hair cells attributable to the motor protein prestin(2,3). Thus outer hair cells contract and elongate in synchrony with the sound-evoked receptor potential(4,5). But problems arise with this mechanism at high frequencies, where the periodic component of the receptor potential will be attenuated by the membrane time constant. On the basis of work in non-mammalian vertebrates, force generation by the hair bundles has been proposed as an alternative means of boosting the mechanical stimulus(6,7). Here we show that hair bundles of mammalian outer hair cells can also produce force on a submillisecond timescale linked to adaptation of the mechano-transducer channels. Because the bundle motor may ultimately be limited by the deactivation rate of the channels, it could theoretically operate at high frequencies. Our results show the existence of another force generator in outer hair cells that may participate in cochlear amplification.

In the first step in auditory transduction, sound-induced vibrations of the stereociliary bundles on the sensory hair cells are converted into electrical signals by opening of mechanotransducer channels. Faithful transduction and hence auditory performance will be limited by the kinetic properties of these channels. We have measured the time course of mechanotransducer currents in turtle and rat auditory hair cells during rapid deflections of the hair bundle. Current activation in the turtle had a time constant that decreased 10-fold with stimulus amplitude to a limiting value of ∼50 μs. Lowering the external Ca 2+ concentration slowed both activation and adaptation time constants. Similar effects were seen in hair cells tuned to low and high frequencies, but the overall kinetics was slower in low-frequency cells. In rat outer hair cells, the time courses of both activation and adaptation were at least 10-fold faster. Although activation kinetics was too fast to characterize accurately, the adaptation time constants in the rat, like the turtle, were Ca 2+ dependent and faster in hair cells tuned to higher frequencies. The results imply that mechanotransducer channels operate similarly in turtle and rat but are faster in the mammal to accommodate its higher frequency range of hearing. We suggest that the kinetics of channel activation and adaptation imposes a bandpass filter on transduction, with a center frequency matched to the frequencies detected by the hair cell, which may improve the signal-to-noise ratio near threshold.

Hair cells in the vertebrate cochlea are arranged tonotopically with their characteristic frequency (CF), the sound frequency to which they are most sensitive, changing systematically with position. Single mechanotransducer channels of hair cells were characterized at different locations in the turtle cochlea. In 2.8 mM external Ca2+, the channel's chord conductance was 118 pS (range 80–163 pS), which nearly doubled (range 149–300 pS) on reducing Ca2+ to 50 μM. In both Ca2+ concentrations, the conductance was positively correlated with hair cell CF. Variation in channel conductance can largely explain the increases in size of the macroscopic transducer current and speed of adaptation with CF. It suggests diversity of transducer channel structure or environment along the cochlea that may be an important element of its tonotopic organization.

In the first step in auditory transduction, vibrations of the hair-cell stereociliary bundle are converted into an electrical signal by the gating of the mechanotransducer channels. Faithful transduction and hence cochlear performance will be limited by, among other things, the kinetic properties of these channels. We have measured the time course of mechanotransducer currents in turtle auditory hair cells during rapid deflections of the hair bundle. The current onset had a principal time constant that decreased 10-fold with stimulus amplitude up to saturation to a limiting value of similar to 40 microseconds. Lowering the external calcium concentration from 2.8 to 0.05 mM slowed both activation and adaptation time constants. Similar effects were seen in hair cells tuned to low and high frequencies, but the overall kinetics were slower in low frequency cells, which argues that transducer kinetics may be matched to the range of frequencies processed by the hair cell. The results suggest that calcium ions, along with bundle displacement, act directly on the mechanotransducer channels to modulate their probability of opening.

Sound stimuli vibrate the hair bundles on auditory hair cells, but the resulting motion attributable to the mechanical stimulus may be modified by forces intrinsic to the bundle, which drive it actively. One category of active hair bundle motion has properties similar to fast adaptation of the mechanotransducer channels and is explicable if gating of the channels contributes significantly to the mechanics of the hair bundle. To explore this mechanism, we measured hair bundle compliance in turtle auditory hair cells under different conditions that alter the activation range of the channel. Force-displacement relationships were nonlinear, possessing a maximum slope compliance when approximately one-half of the transducer channels were open. When the external calcium concentration was reduced from 2.8 to 0.25 mm, the position of maximum compliance was shifted negative, reflecting a comparable shift in the transducer channel activation curve. Assuming that the nonlinearity represents the compliance attributable to channel gating, a single-channel gating force of 0.25 pN was calculated. By comparing bundle displacements with depolarization with and without an attached flexible fiber, the force contributed by each channel was independently estimated as 0.47 pN. These results are consistent with fast active bundle movements resulting from changes in mechanotransducer channel gating. However, several observations revealed additional components of hair bundle motion, with slower kinetics and opposite polarity to the fast movement but also linked to transducer adaptation. This finding argues for multiple mechanisms for controlling hair bundle position in auditory hair cells.

During transduction in auditory hair cells, hair bundle deflection opens mechanotransducer channels that subsequently reclose or adapt to maintained stimuli, a major component of the adaptation occurring on a submillisecond time scale. Using a photodiode imaging technique, we measured hair bundle motion in voltage-clamped turtle hair cells to search for a mechanical correlate of fast adaptation. Excitatory force steps imposed by a flexible glass fiber attached to the bundle caused an initial movement toward the kinocilium, followed by a fast recoil equivalent to bundle stiffening. The recoil had a time course identical to adaptation of the transducer current, and like adaptation, was most prominent for small stimuli, was slowed by reducing extracellular calcium, and varied with hair cell resonant frequency. In free-standing hair bundles, depolarizations positive to 0 mV evoked an outward current attributable to opening of transducer channels, which was accompanied by a sustained bundle deflection toward the kinocilium. Both processes were sensitive to external calcium concentration and were abolished by blocking the transducer channels with dihydrostreptomycin. The similarity in properties of fast adaptation and the associated bundle motion indicates the operation of a rapid calcium-sensitive force generator linked to the gating of the transducer channels. This force generator may permit stimulus amplification during transduction in auditory hair cells.

Annals of the New York Academy of SciencesVolume 656, Issue 1 p. 1-11 The Hair Cell's Mechanoelectrical Transducer Channela R. FETTIPLACE, R. FETTIPLACE Department of Neurophysiology University of Wisconsin Medical School 273 Medical Sciences Building Madison, Wisconsin 53706Search for more papers by this authorA. C. CRAWFORD, A. C. CRAWFORD Physiological Laboratory Cambridge CB2 3EG, United KingdomSearch for more papers by this authorM. G. EVANS, M. G. EVANS Physiological Laboratory Cambridge CB2 3EG, United KingdomSearch for more papers by this author R. FETTIPLACE, R. FETTIPLACE Department of Neurophysiology University of Wisconsin Medical School 273 Medical Sciences Building Madison, Wisconsin 53706Search for more papers by this authorA. C. CRAWFORD, A. C. CRAWFORD Physiological Laboratory Cambridge CB2 3EG, United KingdomSearch for more papers by this authorM. G. EVANS, M. G. EVANS Physiological Laboratory Cambridge CB2 3EG, United KingdomSearch for more papers by this author First published: May 1992 https://doi.org/10.1111/j.1749-6632.1992.tb25196.xCitations: 18 a This work was supported by grants from the Medical Research Council and Royal Society. AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume656, Issue1Sensing and Controlling Motion: Vestibular and Sensorimotor FunctionMay 1992Pages 1-11 RelatedInformation

1. Mechano‐electrical transducer currents evoked by deflections of the hair bundle were recorded in turtle isolated hair cells under whole‐cell voltage clamp. The outcome of perfusing with solutions of reduced Ca2+ concentration was investigated. 2. The transducer current was roughly doubled by lowering the concentration of divalent cations from normal (2.2 mM‐Mg2+, 2.8 mM‐Ca2+) to 0 Mg2+, 0.5 mM‐Ca2+. No significant effects on the current's kinetics or reversal potential, or on the current‐displacement relationship, were noted. 3. If the Ca2+ concentration was lowered to 50 microM (with no Mg2+), there was about a threefold increase in the maximum current but other changes, including loss of adaptation and a decreased slope and negative shift in the current‐displacement relationship, were also observed. As a result, more than half the peak transducer current became activated at the resting position of the hair bundle compared to about a tenth in the control solution. 4. The extra changes manifest during perfusion with 50 microM‐Ca2+ had also been seen when the cell was held at positive potentials near the Ca2+ equilibrium potential. This supports the view that some consequences of reduced external Ca2+ stem from a decline in its intracellular concentration. 5. With 20 microM‐Ca2+, a standing inward current developed and the cell became unresponsive to mechanical stimuli, which may be explained by the transducer channels being fully activated at the resting position of the bundle. 6. The results are interpreted in terms of a dual action of Ca2+: an external block of the transducer channel which reduces the maximum current, and an intracellular effect on the position and slope of the current‐displacement relationship; the latter effect can be modelled by internal Ca2+ stabilizing one of the closed states of the channel. 7. During perfusion with 1 microM‐Ca2+, the holding current transiently increased but then returned to near its control level. There was a concomitant irreversible loss of sensitivity to hair bundle displacements which we suggest is due to rupture of the mechanical linkages to the transducer channel. 8. Following treatment with 1 microM‐Ca2+, single‐channel currents with an amplitude of ‐9 pA at ‐85 mV were sometimes visible in the whole‐cell recording. The probability of such channels being open could be modulated by small deflections of the hair bundle which indicates that they may be the mechano‐electrical transducer channels or conductance about 100 pS. 9. Open‐ and closed‐time distributions for the channel were fitted by single exponentials, the mean open time at rest being approximately 1 ms. The mean open time was increased and the mean closed time decreased for movements of the hair bundle towards the kinocilium.(ABSTRACT TRUNCATED AT 400 WORDS)

If a maintained displacement is imposed upon the ciliary bundle of a hair cell, many of the transducer channels that initially open in response to the stimulus eventually close. This process of adaptation of the transducer current has been investigated in several preparations, by delivering test steps to the bundle following a conditioning step to induce adaptation. The results of such experiments have shown that even after the transducer current has declined extensively from its initial peak value, the transducer channels can still be re-opened by further displacement of the cell’s ciliary bundle towards the kinocilium (Corey & Hudspeth. 1983a,b; Eatock, Corey & Hudspeth, 1987; Assad, Hacohen & Corey, 1989; Crawford, Evans & Fettiplace, 1989). There is therefore no evidence that transducer channels enter a true inactivated state.

1. Transducer currents were recorded in turtle cochlear hair cells during mechanical stimulation of the hair bundle. The currents were measured under whole‐cell voltage clamp in isolated cells that were firmly stuck to the floor of the recording chamber. 2. Stimuli were calibrated by projecting the image of the hair bundle onto a rapidly scanned 128 photodiode array. This technique showed that, while the cell body was immobilized, the tip of the bundle would follow faithfully the motion of an attached glass probe up to frequencies of more than 1 kHz. 3. The relationship between inward transducer current and bundle displacement was sigmoidal. Maximum currents of 200‐400 pA were observed for deflections of the tip of the bundle of 0.5 microns, equivalent to rotating the bundle by about 5 deg. 4. In response to a step deflection of the bundle, the current developed with a time constant (about 0.4 ms for small stimuli) that decreased with the size of displacement. This suggests that the onset of the current was limited by the gating kinetics of the transduction channel. The onset time course was slowed about fourfold for a 20 degrees C drop in temperature. 5. For small maintained displacements, the current relaxed to about a quarter of the peak level with a time constant of 3‐5 ms. This adaptation was associated with a shift of the current‐displacement relationship in the direction of the stimulus. The rate and extent of adaptation were decreased by lowering external Ca2+. 6. Adaptation was strongly voltage sensitive, and was abolished at holding potentials positive to the reversal potential of the transducer current of about 0 mV. It was also diminished by loading cells with 10 mM of the Ca2+ chelator BAPTA. These observations suggest that adaptation may be partly controlled by influx of Ca2+ through the transducer channels. 7. Removal of adaptation produced asymmetric responses, with fast onsets but slow decays following return of the bundle to its resting position; the offset time course depended on both the magnitude and duration of the prior displacement. 8. In some experiments, hair bundles were deflected with a flexible glass fibre whose motion was monitored using a dual photodiode arrangement. Positive holding potentials abolished adaptation of the transducer currents, but had no influence on the time course of motion of the fibre. We have no evidence therefore that adaptation is caused by a mechanical reorganization within the bundle.

The electrical and mechanical properties of single hair cells from the turtle's cochlea were examined to investigate the basis of their electrical resonance. Receptor potentials were measured with intracellular micropipettes in the isolated basilar papilla. At the onset and termination of a step displacement of the ciliary bundle the receptor potential showed a damped oscillation reflecting the frequency selectivity of the cell. Resonance frequencies increased systematically from apex to base of the cochlea. Similar oscillations could be elicited by a current step injected through the recording electrode. Solitary hair cells enzymatically isolated from the papilla were investigated with the tight-seal whole-cell recording method. Cells retained their properties in response to current steps and had resonance frequencies between 10 and 350 Hz. In voltage clamp such cells displayed a large outward K+ current and an inward Ca2+ current both activated by depolarization from the resting potential. The relaxation time constant of the K+ current was inversely correlated with the resonance frequency of the cell, varying from 150 ms in the lowest frequency cells to less than 1 ms in the highest ones. It is argued that variation in the kinetics of this current is the major factor responsible for the range of resonance frequencies. In preparations of the isolated papilla a flexible glass fibre, attached to the tip of a ciliary bundle, was used to deliver constant force steps to the bundle and to monitor its displacement. Receptor potentials were simultaneously recorded. At the beginning and end of a force step towards the kinocilium, the bundle vibrated at a frequency which coincided with the electrical resonance frequency of the cell.(ABSTRACT TRUNCATED AT 250 WORDS)

1. Intracellular recordings were made from single cochlear hair cells in the isolated half‐head of the turtle. The electrical responses of the cells were recorded under two conditions: (a) when the ear was stimulated with low‐intensity tones of different frequencies and (b) when current steps were injected through the intracellular electrode. The aim of the experiments was to evaluate the extent to which the cochlea's frequency selectivity could be accounted for by the electrical properties of the hair cells.2. At low levels of acoustic stimulation, the amplitude of the hair cell's receptor potential was proportional to sound pressure. The linear tuning curve, which is defined as the sensitivity of the cell as a function of frequency when the cell is operating in its linear range, was measured for a number of hair cells with characteristic frequencies from 86 Hz to 425 Hz.3. A rectangular current passed into a hair cell elicited a membrane potential change consisting of a damped oscillation superimposed on a step. Small currents produced symmetrical oscillations at the beginning and end of the pulse. Larger currents increased the initial ringing frequency if depolarizing and decreased it if hyperpolarizing.4. For small currents the frequency of the oscillations and the quality factor (Q) of the electrical resonance derived from the decay of the oscillations were close to the characteristic frequency and Q of the hair‐cell linear tuning curve obtained from sound presentations.5. The hair cell's membrane potential change to small‐current pulses or low‐intensity tone bursts could be largely described by representing the hair cell as a simple electrical resonator consisting of an inductance, resistor and capacitor.6. When step displacements of 29‐250 nm were applied to a micropipette, placed just outside a hair cell in the basilar papilla, an initial periodic firing of impulses could be recorded from single fibres in the auditory nerve. Currents of up to 1 nA, injected through the same micropipette, failed to produce any change in the auditory nerve discharge. The experiment demonstrates that current injection does not produce gross movements of the electrode tip.7. The contribution of the electrical resonance to hair‐cell tuning was assessed by dividing the linear tuning curve by the cell's impedance as a function of frequency. The procedure assumes that the electrical resonance is independent of other filtering stages, and on this assumption the resonance can account for the tip of the acoustical tuning curve.8. The residual filter produced by the division was broad; it exhibited a high‐frequency roll‐off with a corner frequency at 500‐600 Hz, similar in all cells, and a low‐frequency roll‐off, with a corner frequency from 30 to 350 Hz which varied from cell to cell but was uncorrelated with the characteristic frequency of the cell.9. The phase of the receptor potential relative to the sound pressure at the tympanum was measured in ten cells. For low intensities the phase characteristic was independent of the sound pressure. At low frequencies the receptor potential led the sound by 270‐360°, and in the region of the characteristic frequency there was an abrupt phase lag of 90‐180°; the abruptness of the phase change depended upon the Q of the cell.10. The calculated phase shift of the electrical resonator as a function of frequency was subtracted from the phase characteristic of the receptor potential. The subtraction removed the sharp phase transition around the characteristic frequency, and in this frequency region the residual phase after subtraction was approximately constant at +180°. This is consistent with the idea that the hair cells depolarize in response to displacements of the basilar membrane towards the scala vestibuli. The high‐frequency region of the residual phase characteristic was similar in all cells.11. It is concluded that each hair cell contains its own electrical resonance mechanism which accounts for most of the frequency selectivity of the receptor potential. All cells also show evidence of a broad band‐pass filter, the high frequency portion of which may be produced by the action of the middle ear.