Adaptation in auditory hair cells Robert Fettiplace and Anthony J Ricci y

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1 446 Adaptation in auditory hair cells Robert Fettiplace and Anthony J Ricci y The narrow stimulus limits of hair cell transduction, equivalent to a total excursion of about 100 nm 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 2þ 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 2þ 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. Addresses Department of Physiology, University of Wisconsin Medical School, Madison, WI 53706, USA fettiplace@physiology.wisc.edu y Neuroscience Center, Louisiana State University Health Sciences Center, New Orleans, LA 70112, USA This review comes from a themed issue on Sensory systems Edited by Clay Reid and King-Wai Yau /$ see front matter ß 2003 Elsevier Ltd. All rights reserved. DOI /S (03) Abbreviations camp cyclic adenosine monophosphate CF characteristic frequency EP endocochlear potential INAD Inactivation No After-potential D MET mechanoelectrical transducer OHC outer hair cell p o probability of opening of mechanoelectrical transducer channels p o -x relationship between p o and hair bundle displacement s A adaptation time constant x hair bundle displacement Introduction Hair cells are the sensory receptors of the inner ear that convert sound-induced vibrations of the cochlear partition into electrical signals. Mechanoelectrical transduction occurs in the hair bundle, which when moved towards its tallest edge opens mechanically gated ion channels near the tips of the component stereocilia [1]. This allows an influx of K þ and Ca 2þ ions that depolarize the hair cell. The current hypothesis regarding transduction is that deflection of the stereocilia exerts tension on the tip links (Figure 1) that transmit force to the mechanoelectrical transducer (MET) channel through elastic elements that are referred to as gating springs [2]. The double-helical structure of the tip link suggests that it is not the gating spring [3], and the molecular identity of the hair cell MET channel is currently unknown [4]. The probability of opening (p o ) of the MET channels is modulated by hair bundle displacement over a maximal excursion of nm, less than the diameter of a stereocilium. To keep them within a narrow operating range and preserve high sensitivity for extrinsic stimuli, the hair cell MET channels are subject to multiple Ca 2þ -controlled mechanisms of adaptation [5]. This review summarizes recent work on adaptation, including its connection to active hair bundle motion, the contribution of unconventional myosins, and a possible role in cochlear frequency selectivity. Multiple mechanisms of adaptation During a maintained displacement of the hair bundle (x), adaptation appears as a decline in the MET channel p o. This decline reflects a translation of the p o -x relationship along the displacement axis in the direction of the stimulus. At least two adaptation mechanisms can be distinguished on the basis of their different kinetics and mechanical correlates. Fast adaptation in turtle cochlear hair cells has a time constant (t A ) of ms [6,7]. Slow adaptation, first reported in frog saccular cells, has a t A of ms [8,9]. However, both fast and slow mechanisms are now known to coexist in the same hair cell [10,11,12]. The different balance of the two components in turtles and frogs may be due to the fact that turtle hair cells, being auditory, are exposed to higher stimulation frequencies than frog vestibular hair cells. The speed of adaptation may therefore be matched to the frequencies to which the cell is exposed. Consistent with this idea, fast adaptation is most conspicuous in mammalian cochlear hair cells with a t A ¼ 4ms[13]. Moreover, t A in the turtle varies with hair cell characteristic frequency (CF), and is faster in the cells tuned to higher frequencies: the corner frequency (1/2pt A ) of the high-pass filter imposed by fast adaptation is approximately two-thirds of the CF [14]. This suggests that fast adaptation may play some part in hair cell frequency selectivity (the ability of the cell to distinguish different frequency components in the stimulus) [7,14]. Further support for this notion comes from the observation that in physiological Ca 2þ concentrations, fast adaptation can display resonance at frequencies in the turtle s auditory range (Figure 1; [14]). Both types of adaptation are regulated by Ca 2þ that enters the stereocilia through highly Ca 2þ -permeable MET

2 Adaptation in auditory hair cells Fettiplace and Ricci 447 Figure 1 (a) (b) T Myosin Tip link Ca 2+ Out MET channels Myosin Closed C Open O Adapted Ca 2+ C In (c) 2.8 mm Ca 2+ Rootlet P o 0.07 mm Ca ms Current Opinion in Neurobiology Sites and action of hair cell adaptation (a) Structural components of the stereocilia associated with transduction and adaptation, showing the electron dense plaques that represent sub-membranous protein complexes. Rotation towards the taller stereocilium exerts force on the tip link and opens MET channels at the stereociliary tip. Tension in the tip link may be adjusted adaptively by myosin arrays (1c, 7a or 15) at either end of the tip link; for example, myosin-1c in the upper plaque tensions the link by climbing up towards the barbed end of the actin filaments. Stereociliary position could also be influenced by the stiffness of the rootlets into the hair cell apex. (b) Hair bundle displacement tensions the tip link (T) which extends the internal and external gating springs causing the channel to go from the closed (C) to the open (O) configuration. Ca 2þ entering the stereocilium through the open channel binds at the inner face of the channel and shuts it. This generates force by increasing tension in the gating springs. (c) Following a bundle deflection (top), the channel opens rapidly then recloses adaptively in a high concentration of 2.8 mm Ca 2þ. Change in p o is plotted against time. When the extracellular Ca 2þ concentration is reduced to 0.07 mm Ca 2þ, a more realistic physiological value closer to that in cochlear endolymph, the adaptation becomes oscillatory at 77 Hz. The resonant frequency of the MET channel varies with hair cell CF. channels [8,15,16]. Fast adaptation probably requires a direct interaction of Ca 2þ with the MET channels to modulate their probability of opening [14,15,17].Onthe basis of the effects of intracellular calcium buffers, the distance Ca 2þ diffuses to its target is estimated as short, nm from the mouth of the channel [14]. Its resulting action occurs in well under a millisecond [16]. Furthermore, Ca 2þ can alter the time constant of channel activation as well as adaptation [18], arguing that it is intimately linked with channel gating. In contrast, slow adaptation is regarded as an input control, in which a Ca 2þ dependent motor controls tension in the elastic elements in series with the channel [9,19].Thereisgood evidence to implicate myosin-1c as the motor that drives slow adaptation in vestibular hair cells [11 ]. One or both phases of adaptation could be mediated through an interaction with calmodulin [20], which is present at the tips of the stereocilia [21] where it interacts with myosin-1c [22]. Besides the two Ca 2þ -driven mechanisms, other pathways may modulate the MET channel s operating range. For example, cyclic adenosine monophosphate (camp) shifts the p o -x relation along the displacement axis in the positive direction, with no affect on fast adaptation [6].ThecAMPeffectmaybemediated through phosphorylation of the MET channel or the myosin motor by protein kinase A.

3 448 Sensory systems Mechanical correlates of adaptation The distinction between fast (channel) adaptation and slow (myosin motor) adaptation is supported by measurements of hair bundle motion during electrical or mechanical hair cell stimulation. The connection between these two types of stimuli is their effect on the intracellular Ca 2þ concentration. Both displacement of the hair bundle towards its shorter edge (closing the MET channels) and depolarization towards the Ca 2þ equilibrium potential (lowering the electrical driving force on Ca 2þ ) reduce Ca 2þ influx. The evoked movements can be classified on the basis of their kinetics, which match those of fast and slow adaptation. The fast active response is complete in a few milliseconds [23 25], but the slow response can extend from 10 to 100 ms [9,19]. The time constant of the fast mechanical response, like that of fast adaptation, varies with hair cell CF and is faster in cells tuned to higher frequencies [23]. It is also possible to distinguish the two mechanisms by their polarity. For large depolarizations that take the membrane potential near the Ca 2þ equilibrium potential, the fast movement is in the positive direction (towards the taller edge of the bundle), whereas the slow movement is in the negative direction. As with adaptation, both components are observable in the same cell [26 ]. An interesting but unexplained observation is that the speed and polarity of the movement depend on the absolute bundle position: steady displacement of the bundle towards its taller edge transforms a fast positive motion into a slower negative one [26 ]. A prediction of the gating spring model of transduction is that as the channel opens there is a decrease in hair bundle stiffness [2], and this is confirmed experimentally [2,26,27, 28]. Ca 2þ interaction with the channel to modulate its p o will therefore cause the bundle to move, connecting fast adaptation to bundle motion [23,28]. In terms of polarity, a positive bundle deflection opens the MET channels and increases Ca 2þ concentration, which recloses the channels and causes negative recoil. In contrast, the increase in Ca 2þ produced by a positive bundle deflection detaches the myosin from the actin core of the stereocilium. This allows the myosin and its attachment to the tip link to slip down the stereocilium, which leads to further positive displacement of the bundle [9]. Evidence for the range of movement of the tip-link s upper attachment point comes from the observation that if the tip links are severed with BAPTA (1,2-bis[o-Aminophenoxy]ethane-N,N,N,N -tetraacetic acid), the electron dense plaque (Figure 1) climbs nm closer to the stereociliary tip [29]. An unconventional myosin as the adaptation motor Five unconventional non-muscle myosins have been localized to hair cells: myosin-1c, 3, 6, 7a and 15 [30,31,32].Of these, the prime contenders for the motor that powers slow adaptation are either myosin-1c [11 ] or myosin 7a [33 ]. Myosin-1c is concentrated in frog hair bundles at the two ends of the tip link: not only in the electron-dense plaque marking its upper attachment point but also most conspicuously at the tip of the stereocilium [34,35]. Convincing evidence for the role of myosin-1c in slow adaptation has come from modifying the ATP-binding site to confer susceptibility to inhibition by certain ADP analogs [36]. Expression of the mutated myosin-1c in mouse utricular hair cells was then shown to render slow adaptation sensitive to blockade by the ADP analogs introduced through the recording pipette, whereas it did not affect fast adaptation [11 ]. Although myosin-7a is distributed along the entire length of the stereocilia [30], its mutation in Shaker1 mice causes a substantial positive shift of the p o -x relation along the displacement axis so that the MET channels are no longer poised to open at the bundle s resting position [33 ]. Thus, myosin-7a may also contribute to adaptation, but how it collaborates with myosin-1c in optimizing transduction is not well understood. Do the two myosin isoforms generate force in the same direction or do they operate in an opposable push-pull manner? How are they controlled by changes in intracellular Ca 2þ concentration? To relieve tip link tension Ca 2þ must cause detachment of the myosin head from actin and its slippage down the stereocilia [5,37], rather than the usual initiation of the power stroke, which would drive the myosin up the stereocilia and therefore increase tip link tension. Myosin-6 is anomalous in tracking backwards along the actin filament [38], and would be more suited as the motor at the top of the tip link. At this point less is known about myosin-3 and myosin-15 so their roles in transduction cannot be properly assessed. Myosin-3 is especially interesting because its homolog is present in Drosophila photoreceptors where it associates with the PDZ scaffolding protein INAD (Inactivation No Afterpotential D) that also binds to the photoreceptor transducer channel [39]. INAD is also present in the vertebrate cochlea [31 ]. The unconventional myosins may perform other cellular functions besides adaptation, including ferrying components of the transduction machinery to their appropriate location [40 ]. Furthermore, mutations in several of the myosins are accompanied by loss of the hair bundle s structural integrity that results in deafness [41,42]. The reverse motor action of myosin-6 [38] may enable it to retrieve proteins transported to the stereociliary tips by other forward-acting myosins. Roles of adaptation mechanisms A reason for having multiple adaptation mechanisms is that the slower mechanism has a wider dynamic range to orient the bundle to a location where fast feedback control of the channels is effective. Thus, fast adaptation may tune the channel for small displacements around a resting position that is continually readjusted by slow adaptation. Fast adaptation could theoretically participate in auditory frequency selectivity by filtering the MET current [14] or by generating fast hair bundle movements

4 Adaptation in auditory hair cells Fettiplace and Ricci 449 that amplify the mechanical input. Hair bundles in the frog saccule have the ability to mechanically amplify a signal leading to spontaneous oscillations. The oscillations occur at low frequencies (5 50 Hz) and it has been hypothesized that they are driven by the slow myosin motor, biasing the displacement-force relation of the hair bundle into a region of negative slope [43 ]. In contrast, active bundle movements produced by Ca 2þ binding to the MET channels can in principle occur at kilohertz frequencies, within the mammalian auditory range [17]. Furthermore, trans-epithelial electrical stimulation of the isolated frog saccule experimentally evokes hair bundle oscillations at frequencies of up to 1 khz [44 ]. There is also in vivo evidence for active hair bundle motion at more than 1 khz in the lizard hearing organ [45 ]. However, it remains to be seen whether or not active motion of the outer hair-cell bundles can generate sufficient force to produce amplification in the intact mammalian cochlea [7]. Tuning of the MET current in turtle auditory hair cells does not require concomitant active bundle motion because it occurs when the bundle is displaced with a rigid stimulating probe (Figure 1; [14]). This suggests that the energy associated with channel gating does not need to move the hair bundle in order to elicit oscillations in the current. The variation in resonant frequency ( Hz), which is within the turtle s auditory range, may therefore involve differences in the MET channel. Several mechanisms for these differences have been proposed. The adaptation rate or resonant frequency increases with a higher stereociliary Ca 2þ concentration, which could be brought about either by increasing the channel sca 2þ permeability with CF or by increasing the conductance of the MET channels or their number per stereocilium. The evidence for this mechanism is that the maximum MET current increases with CF, and the rate of adaptation at a given CF varies with the magnitude of the current [6,14]. It has been recently shown that the channel sca 2þ permeability does not alter with CF [46 ]. However, there is evidence for a tonotopic variation in channel kinetics derived from noise analysis of the MET current [46 ], and from measurements of the time course of current activation [18]. An alternate view, formed on the basis of modeling hair bundle amplification, is that both fast and slow adaptation processes combine to produce oscillations at a frequency determined by hair bundle geometry and intracellular Ca 2þ dynamics [47]. Amplification and tuning in the mammalian cochlea In the mammalian cochlea, the outer hair cells (OHC) are responsible for generating active mechanical amplification that provides the compressive non-linearity and frequency selectivity of the basilar membrane [48]. The ability of OHCs to elongate and shorten in response to changes in membrane potential is thought to supply the mechanical energy for the process [49]. Prestin has been identified as the protein responsible for this somatic motility [50], and it is concentrated in the lateral wall of the OHC [51]. The voltage sensitivity of prestin is endowed by the intracellular binding of small anions such as Cl [52 ]. Targeted deletion of prestin in mice results in an elevated threshold, reduced tuning, and loss of outer hair cell motility [53 ], which led the authors to conclude that somatic motility alone was responsible for the active process. This conclusion can be criticized on the grounds that any feedback process involving multiple elements would be compromised if one element were eliminated. For example, abolition of the endocochlear potential (EP) by treatment with the diuretic furosemide reduces amplification about 30-fold on average [54], even though the EP is not the source of the active process. Loss of the EP will approximately halve the MET current. With regards to the same argument, halving of electromotility in prestin heterozygotes [53 ] should have produced greater than the twofold elevation in cochlear thresholds if somatic motility were the sole source of the active process. Furthermore, although OHC motility may supply the energy for amplification, there is no evidence that it is intrinsically frequency selective. For cochlear models to produce realistically sharp tuning of the basilar membrane, the mechanical feedback from the outer hair cells must occur in a frequency-selective manner to ensure it supplies force at the appropriate phase of basilar membrane vibration [55 58]. The required tuning has been ascribed to a mechanical resonance in the tectorial membrane [56,58,59], but it could equally reside in the MET channels. If the channels in mammalian OHCs operate similarly to those in turtle hair cells, the transducer currents and any associated active hair bundle motion will be tuned over a frequency range imparted by variations in the speed of fast adaptation [23]. Conclusions Over the past five years, evidence has emerged for at least two distinct Ca 2þ -mediated mechanisms of hair cell transducer adaptation varying in speed, range and function. A fast mechanism directly affects MET channel gating, whereas a slower one regulates the mechanical stimulus through the action of one or more unconventional myosins. Further insights into the roles of the different myosins may come from the elucidation of their subcellular localization using post-embedding immunogold techniques, or their modification and deletion in transgenic animals. The fast mechanism confers tuning on the MET channels and may be an important factor in cochlear frequency selectivity. Because adaptation shifts the hair cell s operating point, it may have an ancillary role in constantly adjusting and optimizing the signal-to-noise ratio of transduction [60]. Cloning the MET channel should illuminate how the channel interacts with Ca 2þ and other subcellular components including myosins. It may also reveal the existence of multiple channel isoforms with Ca 2þ affinity or kinetics specialized for operation in different frequency ranges. However, further

5 450 Sensory systems electrophysiological recordings in the mammalian cochlea will be needed to settle whether or not fast adaptation is present in outer hair cells and whether or not it is sufficiently fast to participate in the active process. Evidence that this may be the case was recently obtained in rat OHCs, in which transducer currents exhibited fast Ca 2þ - dependent adaptation with a t A of less than 0.2 ms, faster than any seen in turtle auditory hair cells [61 ]. Acknowledgements Work in the authors laboratories was supported by the National Institute on Deafness and Other Communicative Disorders, grants RO1 DC (to R Fettiplace) and RO1 DC (to AJ Ricci). We thank C Hackney for her comments on the manuscript and N Cooper for pointing out the amplification discrepancy in the prestin heterozygotes. References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest 1. Hudspeth AJ: How the ear s works work. Nature 1999, 341: Howard J, Hudspeth AJ: Compliance of the hair bundle associated with gating of mechanoelectrical transduction channels in the bullfrog s saccular hair cell. Neuron 1988, 1: Kachar B, Parakkal M, Kurc M, Zhao Y, Gillespie PG: Highresolution structure of hair-cell tip links. Proc Natl Acad Sci USA 2000, 97: Strassmaier M, Gillepsie PG: The hair cell s transduction channel. Curr Opin Neurobiol 2002, 12: Eatock RA: Adaptation in hair cells. Annu Rev Neurosci 2000, 23: Ricci AJ, Fettiplace R: The effects of calcium buffering and cyclic AMP on mechano-electrical transduction in turtle auditory hair cells. J Physiol 1997, 501: Fettiplace R, Ricci AJ, Hackney CM: Clues to the cochlear amplifier from the turtle ear. Trends Neurosci 2001, 24: Assad JA, Hacohen N, Corey DP: Voltage dependence of adaptation and active bundle movement in bullfrog saccular hair cells. Proc Natl Acad Sci USA 1989, 86: Assad JA, Corey DP: An active motor model for adaptation by vertebrate hair cells. J Neurosci 1992, 12: Wu YC, Ricci AJ, Fettiplace R: Two components of transducer adaptation in auditory hair cells. J Neurophysiol 1999, 82: Holt JR, Gillespie SK, Provance DW, Shah K, Shokat KM, Corey DP, Mercer JA, Gillespie PG: A chemical-genetic strategy implicates myosin-1c in adaptation by hair cells. Cell 2002, 108: The authors present unequivocal evidence of a role for myosin-1c in slow adaptation in mouse vestibular hair cells. The work uses a strikingly novel pharmacological technique of mutating the receptor (ATPase) site on myosin-1c to render it susceptible to blockade by engineered adenosine diphosphate analogs. 12. Eatock RA, Hurley KM, Vollrath M: Mechanoelectrical and voltage-gated ion channels in mammalian avestibular hair cells. Audiol Neurootol 2002, 7: Kros CJ, Rüsch A, Richardson GP: Mechano-electrical transducer currents in hair cells of the cultured neonatal mouse cochlea. Proc R Soc Lond B Biol Sci 1992, 249: Ricci AJ, Wu Y-C, Fettiplace R: The endogenous Ca 2R buffer and the time course of transducer adaptation in auditory hair cells. J Neurosci 1998, 18: Crawford AC, Evans MG, Fettiplace R: Activation and adaptation of transducer currents in turtle hair cells. J Physiol 1989, 419: Ricci AJ, Fettiplace R: Calcium permeation of the turtle hair cell mechanotransducer channel and its relation to the composition of endolymph. J Physiol 1998, 506: Choe Y, Magnasco M, Hudspeth AJ: A model for amplification of hair bundle motion by cyclical binding of Ca 2R to mechanoelectrical transducer channels. Proc Natl Acad Sci USA 1998, 95: Fettiplace R, Crawford AC, Ricci AJ: The effects of calcium on mechanotransducer channel kinetics in auditory hair cells. In Biophysics of the Cochlea: From Molecules to Models. Edited by Gummer AW. Singapore: World Scientific; 2003, Howard J, Hudspeth AJ: Mechanical relaxation of the hair bundle mediates adaptation in mechanoelectrical transduction by the bullfrog s saccular hair cell. Proc Natl Acad Sci USA 1987, 84: Walker RG, Hudspeth AJ: Calmodulin controls adaptation of mechanoelectrical transduction by hair cells of the bullfrog s sacculus. Proc Natl Acad Sci USA 1996, 93: Furness DN, Karkanevatos A, West B, Hackney CM: An immunogold investigation of the distribution of calmodulin in the apex of the cochlear hair cells. Hear Res 2002, 173: Cyr JL, Dumont RA, Gillespie PG: Myosin 1-c interacts with haircell receptors through its calmodulin- binding IQ domains. J Neurosci 2002, 22: Ricci AJ, Crawford AC, Fettiplace R: Active hair bundle motion linked to fast transducer adaptation in auditory hair cells. J Neurosci 2000, 20: Crawford AC, Fettiplace R: The mechanical properties of ciliary bundles of turtle cochlear hair cells. J Physiol 1985,364: Benser ME, Marquis RE, Hudspeth AJ: Rapid, active hair bundle movements inhair cellsfrom the bullfrog s sacculus. J Neurosci 1996, 16: Ricci AJ, Crawford AC, Fettiplace R: Mechanisms of active hair bundle motion in auditory hair cells. J Neurosci 2002, 22: This study provides evidence for at least two components of adaptation and their manifestation in active bundle motion in the same hair cell, in which they have characteristic kinetics and polarities. It also contains the surprising observation that by biasing the static position of the hair bundle one of the movements can be reversed in polarity through an as yet unknown mechanism. 27. Russell IJ, Kössl M, Richardson GP: Nonlinear mechanical responses of mouse cochlear hair bundles. Proc R Soc Lond B Biol Sci 1992, 250: van Netten SM, Kros CJ: Gating energies and forces of the mammalian hair cell transducer channel and related hair bundle mechanics. Proc R Soc Lond B Biol Sci 2000, 267: Shepherd GMG, Assad JA, Solc CK, Rock KE, Corey DP: A molecular motor mediating adaptation in bullfrog hair cells. In Biophysics of Hair Cell Sensory Systems. Edited by Duifhuis H, Horst JW, van Dijk P, van Netten SM. Singapore: World Scientific; 1993, Hasson T, Gillespie PG, Garcia JA, MacDonald RB, Zhao Y, Yee AG, Mooseker MS, Corey DP: Unconventional myosins in inner-ear sensory epithelia. J Cell Biol 1997, 137: Walsh T, Walsh V, Vreugde S, Hertzano R, Shahin H, Hsika S, Lee MK, Kanaan M, King M-C, Avraham K: From flies eyes to our ear: mutations in a human class III myosin causes nonsyndromic hearing loss DFNB30. Proc Natl Acad Sci USA 2002, 99: The authors provide the first demonstration of the involvement of myosin III in hearing and its targeting to cochlear hair cells. This class of myosin was previously known to interact with actin filaments and a PDZ scaffolding protein in Drosophila photoreceptors. Here, three mutations of myosin IIIa were shown to underlie a nonsyndromic progressive deafness. 32. Belyantseva IA, Azevedo RB, Fridell RA, Friedman TB, Kachar B: Localization of myosin XVA in hair cells. ARO Abstr 2002,25:157.

6 Adaptation in auditory hair cells Fettiplace and Ricci Kros CJ, Marcotti W, van Netten SM, Self TJ, Libby RT, Brown DM, Richardson GP, Steel KP: Reduced climbing and increased slipping adaptation in cochlear hair cells of mice with Myo7a mutations. Nat Neurosci 2002, 5: The authors use post-genomic experimentation that employs physiological measurements in mouse mutants to elucidate a role for myosin 7a in mechanotransduction in auditory hair cells. For the MET channels to open, the hair bundles in the mutants must be deflected several microns beyond their normal operating range, which suggests that myosin 7a normally positions the bundle for optimal transducer sensitivity. 34. Garcia JA, Yee AG, Gillespie PG, Corey DP: Localization of myosin-ibeta near both ends of tip links in frog saccular hair cells. J Neurosci 1998, 18: Steyger PS, Gillespie PG, Baird RA: Myosin Ibeta is located at tip link anchors in vestibular hair bundles. J Neurosci 1998, 18: Gillespie PG, Gillespie SK, Mercer JA, Shah K, Shokat KM: Engineering of the myosin-ibeta nucleotide-binding pocket to create selective sensitivity to N(6)-modifiedADP analogs. J Biol Chem 1999, 274: Gillespie PG, Corey DP: Myosin and adaptation by hair cells. Neuron 1997, 19: Wells AL, Lin AW, Chen LQ, Saler D, Cain SM, Hasson T, Carraghers BO, Milligan RA, Sweeney HL: Myosin VI is an actinbased motor that moves backwards. Nature 1999, 401: Wes PD, Xu XS, Li H, Chien F, Doberstein SK, Montell C: Termination of phototransduction requires binding of the NINAC myosin III and the PDZ protein INAD. Nat Neurosci 1999, 2: Boëda B, El-Amraoui A, Bahloul A, Goodyear R, Daviet L, Blanchard S, Perfettini I, Fath KR, Shorte S, Reiners J et al.: Myosin VIIa, harmonin and cadherin 23, three Usher I gene products that cooperate to shape the sensory hair bundle. EMBO J 2002, 21: This study uses both yeast two-hybrid and pull-down binding assays to demonstrate interactions among three proteins (myosin 7a, cadherin-23 and a PDZ protein harmonin) that are known to be mutated in Usher type I deaf-blindness syndrome. Equivalent mutations in mice cause defective hair bundle development and result in deafness. The authors conclude that myosin 7a ferries harmonin along the actin core of developing stereocilia, and that the three-protein complex is essential for stereociliary cohesion. 41. Self T, Mahony M, Fleming J, Walsh J, Brown SD, Steele KP: Shaker-1 mutations reveal roles for myosin VIIA in both development and function of cochlear hair cells. Development 1998, 125: Anderson DW, Probst FJ, Belyantsevsa IA, Fridell RA, Beyer L, Martin DM, Wu D, Kachar B, Friedman TB, Raphael Y, Camper S: The motor and tail regions of myosin XV are critical for normal structure and function of auditory and vestibular hair cells. Hum Mol Genet 2000, 12: Martin P, Bozovic D, Choe Y, Hudspeth AJ: Spontaneous oscillations by hair bundles of the bullfrog s sacculus. J Neurosci 2003, 23: The authors present the most detailed account so far of the properties and mechanisms of spontaneous hair bundle oscillations. They demonstrate that they are driven by the slow adaptation motor biasing the displacementforce relation of the hair bundle into a region of negative slope. 44. Bosovic D, Hudspeth AJ: Hair-bundle movements elicited by transepithelial electrical stimulation of hair cells in the sacculus of the bullfrog. Proc Natl Acad Sci USA 2003, 100: The authors describe a single-cell correlate of the electrically evoked otoacoustic emissions that are recorded in most vertebrate classes and used as evidence for ubiquitous active mechanical output from the hair cells. Spontaneous oscillations of saccular hair bundles were modulated at frequencies of up to 1 khz by extracellular electrical stimulation. The specificity of the response was tied to hair cell transduction by showing that it disappeared on blocking the MET channels. 45. Manley GA, Kirk DL, Köppl C, Yates GK: In vivo evidence for a cochlear amplifier in the hair-cell bundle of lizards. Proc Natl Acad Sci USA 2001, 98: Good evidence for active hair bundle motion in vivo comes from the properties of the otoacoustic emissions produced by electrical stimulation of the lizard inner ear. The authors show that because the hair bundles in the lizard hearing organ have two polarities of orientation, the ototacoustic emissions display a characteristic pattern of modulation by sound. 46. Ricci A: Differences in mechano-transducer channel kinetics underlie tonotopic distribution of fast adaptation in auditory hair cells. J Neurophysiol 2002, 87: The authors demonstrate that MET currents in turtle auditory hair cells tuned to high and low frequencies show different sensitivities to channel blockers such as dihydrostreptomycin, and have different open times as assessed by the spectra of the current noise. 47. Vilfan A, Duke T: Two adaptation processes in auditory hair cells together can provide an active amplifier. Biophys J 2003, 85: Robles L, Ruggero MA: Mechanics of the mammalian cochlea. Physiol Rev 2001, 81: Nobili R, Mammano F, Ashmore JF: How well do we understand the cochlea? Trends Neurosci 1998, 21: Zheng J, Shen W, He DZ, Long KB, Madison LD, Dallos P: Prestin is the motor protein of cochlear outer hair cells. Nature 2000, 405: Belyantseva IA, Adler HJ, Curi R, Frolenkov GI, Kachar B: Expression and localization of prestin and the sugar transporter GLUT-5 during development of electromotility in cochlear outer hair cells. J Neurosci 2000, 20:RC Oliver D, He DZZ, Klöcker N, Ludwig J, Schulte U, Waldegger S, Ruppersberg JP, Dallos P, Fakler B: Intracellular anions as the voltage sensor or prestin, the outer hair cell motor protein. Science 2001, 292: The authors found that mutagenesis of many of the amino acids from charged to neutral in the putative membrane domain of the prestin molecule had little or no effect on the voltage sensitivity of the membrane protein. This led them to conclude that the voltage sensor for the outer hair cell motor was a charged particle extrinsic to the protein, and they identified it as arising from the binding of intracellular chloride and bicarbonate ions. 53. Liberman MC, Gao J, He DZZ, Wu X, Jia S, Zuo J: Prestin is required for electromotility of the outer hair cell and for the cochlear amplifier. Nature 2002, 419: The authors found that targeted deletion of prestin in mice abolishes the somatic motility of outer hair cells in vitro and causes a db loss of auditory sensitivity and otoacoustic emissions in vivo without affecting the MET channels. This is strong evidence for prestin being part of the electromechanical feedback loop that underlies cochlear amplifier. 54. Ruggero MA, Rich NC: Furosemide alters organ of Corti mechanics: evidence for feedback of outer hair cells upon the basilar membrane. J Neurosci 1991, 11: Neely ST, Kim DO: An active cochlear model showing sharp tuning and high sensitivity. Hear Res 1983, 9: Nobili R, Mammano F: Biophysics of the cochlea II: stationary nonlinear phenomenology. J Acoust Soc Am 1996,99: Nilsen KE, Russell IJ: The spatial and temporal representation of a tone on the guinea pig basilar membrane. Proc Natl Acad Sci USA 2000, 97: Geisler CD, Sang C: A cochlear model using feed-forward outerhair-cell forces. Hear Res 1995, 86: Gummer AW, Hemmert W, Zenner H-P: Resonant tectorial membrane motion in the inner ear: its crucial role in frequency tuning. Proc Natl Acad Sci USA 1996, 93: Dinklo T, van Netten SM, Marcotti W, Kros CJ: Signal processing by transducer channels in mammalian outer hair cells. In Biophysics of the cochlea: from molecules to models. Edited by Gummer AW. Singapore: World Scientific; 2003, Kennedy HJ, Evans MG, Crawford AC, Fettiplace R: Fast adaptation of mechanoelectrical transducer channels in mammalian cochlear hair cells. Nat Neurosci (Published online DOI: / Nn1089). The authors use the first measurements of OHC transducer currents in animals after the onset of hearing to demonstrate that the MET channels in mammalian cochlear hair cells possess ultra-fast activation and adaptation kinetics.