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Adaptation in auditory hair cells Robert Fettiplace and Anthony J Ricciy

The narrow stimulus limits of transduction, equivalent to deflection of the exerts tension on the tip links a total excursion of about 100 nm at the tip of the hair bundle, (Figure 1) that transmit force to the mechanoelectrical demand tight regulation of the mechanical input to ensure that transducer (MET) channel through elastic elements that the mechanoelectrical transducer (MET) channels operate in are referred to as gating springs [2]. The double-helical their linear range. This control is provided by multiple structure of the tip link suggests that it is not the gating components of Ca2þ–dependent adaptation. A slow mechanism spring [3], and the molecular identity of the hair cell MET limits the mechanical stimulus through the action of one or more channel is currently unknown [4]. The probability of unconventional myosins. There is also a fast, sub-millisecond, opening (po) of the MET channels is modulated by hair Ca2þ regulation of the MET channel, which can generate bundle displacement over a maximal excursion of 100– resonance and confer tuning on transduction. Changing the 200 nm, less than the diameter of a stereocilium. To keep conductance or kinetics of the MET channels can vary their them within a narrow operating range and preserve high resonant frequency. The tuning information conveyed in sensitivity for extrinsic stimuli, the hair cell MET chan- transduction may combine with the somatic motility of outer hair nels are subject to multiple Ca2þ-controlled mechanisms cells to produce an active process that supplies amplification of adaptation [5]. This review summarizes recent work on and augments frequency selectivity in the mammalian . adaptation, including its connection to active hair bundle motion, the contribution of unconventional myosins, and Addresses a possible role in cochlear frequency selectivity. Department of Physiology, University of Wisconsin Medical School, Madison, WI 53706, USA Multiple mechanisms of adaptation e-mail: [email protected] yNeuroscience Center, Louisiana State University Health Sciences During a maintained displacement of the hair bundle (x), Center, New Orleans, LA 70112, USA adaptation appears as a decline in the MET channel po. This decline reflects a translation of the po-x relationship along the displacement axis in the direction of the sti- Current Opinion in Neurobiology 2003, 13:446–451 mulus. At least two adaptation mechanisms can be dis- This review comes from a themed issue on tinguished on the basis of their different kinetics and Sensory systems mechanical correlates. Fast adaptation in turtle cochlear Edited by Clay Reid and King-Wai Yau hair cells has a time constant (tA) of 0.3–5 ms [6,7]. Slow 0959-4388/$ – see front matter adaptation, first reported in frog saccular cells, has a tA of ß 2003 Elsevier Ltd. All rights reserved. 10–100 ms [8,9]. However, both fast and slow mechan- isms are now known to coexist in the same hair cell DOI 10.1016/S0959-4388(03)00094-1 [10,11,12]. The different balance of the two compo- nents in turtles and frogs may be due to the fact that turtle Abbreviations hair cells, being auditory, are exposed to higher stimula- cAMP cyclic adenosine monophosphate tion frequencies than frog vestibular hair cells. The speed CF characteristic frequency of adaptation may therefore be matched to the frequen- EP endocochlear potential INAD Inactivation No After-potential D cies to which the cell is exposed. Consistent with this MET mechanoelectrical transducer idea, fast adaptation is most conspicuous in mammalian OHC outer hair cell cochlear hair cells with a tA ¼ 4ms[13]. Moreover, tA in po probability of opening of mechanoelectrical transducer channels the turtle varies with hair cell characteristic frequency po-x relationship between po and hair bundle displacement (CF), and is faster in the cells tuned to higher frequen- sA adaptation time constant x hair bundle displacement cies: the corner frequency (1/2ptA) of the high-pass filter imposed by fast adaptation is approximately two-thirds of the CF [14]. This suggests that fast adaptation may play Introduction some part in hair cell frequency selectivity (the ability of Hair cells are the sensory receptors of the inner that the cell to distinguish different frequency components in convert sound-induced vibrations of the cochlear partition the stimulus) [7,14]. Further support for this notion comes into electrical signals. Mechanoelectrical transduction from the observation that in physiological Ca2þ concen- occurs in the hair bundle, which when moved towards trations, fast adaptation can display resonance at frequen- its tallest edge opens mechanically gated ion channels cies in the turtle’s auditory range (Figure 1; [14]). near the tips of the component stereocilia [1]. This allows an influx of Kþ and Ca2þ ions that depolarize the hair cell. Both types of adaptation are regulated by Ca2þ that enters The current hypothesis regarding transduction is that the stereocilia through highly Ca2þ-permeable MET

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Figure 1

(a) (b) T

Ca2+ Myosin Out Tip link

In MET channels Myosin Closed C Open O Adapted Ca2+ C

(c)

2.8 mM Ca2+

Rootlet

P o 0.07 mM Ca2+

20 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. Ca2þ 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 2þ deflection (top), the channel opens rapidly then recloses adaptively in a high concentration of 2.8 mM Ca . Change in po is plotted against time. When the extracellular Ca2þ concentration is reduced to 0.07 mM Ca2þ, a more realistic physiological value closer to that in cochlear , 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 evidence to implicate myosin-1c as the motor that drives direct interaction of Ca2þ with the MET channels to slow adaptation in vestibular hair cells [11]. One or both modulate their probability of opening [14,15,17].Onthe phases of adaptation could be mediated through an basis of the effects of intracellular calcium buffers, the interaction with calmodulin [20], which is present at distance Ca2þ diffuses to its target is estimated as short, the tips of the stereocilia [21] where it interacts with 15–35 nm from the mouth of the channel [14]. Its result- myosin-1c [22]. Besides the two Ca2þ-driven mechan- ing action occurs in well under a millisecond [16]. isms, other pathways may modulate the MET channel’s Furthermore, Ca2þ can alter the time constant of channel operating range. For example, cyclic adenosine mono- activation as well as adaptation [18], arguing that it is phosphate (cAMP) shifts the po-x relation along the intimately linked with channel gating. In contrast, slow displacement axis in the positive direction, with no affect adaptation is regarded as an input control, in which a on fast adaptation [6].ThecAMPeffectmaybemediated Ca2þ–dependent motor controls tension in the elastic through phosphorylation of the MET channel or the elements in series with the channel [9,19].Thereisgood myosin motor by protein kinase A. www.current-opinion.com Current Opinion in Neurobiology 2003, 13:446–451 448 Sensory systems

Mechanical correlates of adaptation the two ends of the tip link: not only in the electron-dense The distinction between fast (channel) adaptation and plaque marking its upper attachment point but also most slow (myosin motor) adaptation is supported by measure- conspicuously at the tip of the stereocilium [34,35]. ments of hair bundle motion during electrical or mechan- Convincing evidence for the role of myosin-1c in slow ical hair cell stimulation. The connection between these adaptation has come from modifying the ATP-binding two types of stimuli is their effect on the intracellular site to confer susceptibility to inhibition by certain ADP Ca2þ concentration. Both displacement of the hair bundle analogs [36]. Expression of the mutated myosin-1c in towards its shorter edge (closing the MET channels) and mouse utricular hair cells was then shown to render slow depolarization towards the Ca2þ equilibrium potential adaptation sensitive to blockade by the ADP analogs (lowering the electrical driving force on Ca2þ) reduce introduced through the recording pipette, whereas it Ca2þ influx. The evoked movements can be classified on did not affect fast adaptation [11]. the basis of their kinetics, which match those of fast and slow adaptation. The fast active response is complete in a Although myosin-7a is distributed along the entire length few milliseconds [23–25], but the slow response can of the stereocilia [30], its mutation in Shaker1 mice causes a extend from 10 to 100 ms [9,19]. The time constant of substantial positive shift of the po-x relation along the the fast mechanical response, like that of fast adaptation, displacement axis so that the MET channels are no longer varies with hair cell CF and is faster in cells tuned to poised to open at the bundle’s resting position [33]. Thus, higher frequencies [23]. It is also possible to distinguish myosin-7a may also contribute to adaptation, but how it the two mechanisms by their polarity. For large depolar- collaborates with myosin-1c in optimizing transduction is izations that take the membrane potential near the Ca2þ not well understood. Do the two myosin isoforms generate equilibrium potential, the fast movement is in the positive force in the same direction or do they operate in an direction (towards the taller edge of the bundle), whereas opposable push-pull manner? How are they controlled the slow movement is in the negative direction. As with by changes in intracellular Ca2þ concentration? To relieve adaptation, both components are observable in the same tip link tension Ca2þ must cause detachment of the myosin cell [26]. An interesting but unexplained observation is head from actin and its slippage down the stereocilia [5,37], that the speed and polarity of the movement depend on the rather than the usual initiation of the power stroke, which absolute bundle position: steady displacement of the bun- would drive the myosin up the stereocilia and therefore dle towards its taller edge transforms a fast positive motion increase tip link tension. Myosin-6 is anomalous in track- into a slower negative one [26]. ing backwards along the actin filament [38], and would be more suited as the motor at the top of the tip link. At this A prediction of the gating spring model of transduction is point less is known about myosin-3 and myosin-15 so their that as the channel opens there is a decrease in hair bundle roles in transduction cannotbe properly assessed. Myosin-3 stiffness [2], and this is confirmed experimentally [2,26,27, is especially interesting because its homolog is present in 2þ 28].Ca interaction with the channel to modulate its po Drosophila photoreceptors where it associates with the will therefore cause the bundle to move, connecting fast PDZ scaffolding protein INAD (Inactivation No After- adaptation to bundle motion [23,28]. In terms of polarity, a potential D) that also binds to the photoreceptor transdu- positive bundle deflection opens the MET channels and cer channel [39]. INAD is also present in the vertebrate increases Ca2þ concentration, which recloses the channels cochlea [31]. The unconventional myosins may perform and causes negative recoil. In contrast, the increase in Ca2þ other cellular functions besides adaptation, including fer- produced by a positive bundle deflection detaches the rying components of the transduction machinery to their myosin from the actin core of the stereocilium. This allows appropriate location [40]. Furthermore, mutations in the myosin and its attachment to the tip link to slip down several of the myosins are accompanied by loss of the hair the stereocilium, which leads to further positive displace- bundle’s structural integrity that results in deafness ment of the bundle [9]. Evidence for the range of move- [41,42]. The reverse motor action of myosin-6 [38] may ment of the tip-link’s upper attachment point comes from enable it to retrieve proteins transported to the stereo- the observation that if the tip links are severed with ciliary tips by other forward-acting myosins. BAPTA (1,2-bis[o-Aminophenoxy]ethane-N,N,N’,N’-tet- raacetic acid), the electron dense plaque (Figure 1) climbs Roles of adaptation mechanisms 50–100 nm closer to the stereociliary tip [29]. A reason for having multiple adaptation mechanisms is that the slower mechanism has a wider dynamic range to An unconventional myosin as the orient the bundle to a location where fast feedback adaptation motor control of the channels is effective. Thus, fast adaptation Five unconventional non-muscle myosins have been local- may tune the channel for small displacements around a ized to hair cells: myosin-1c, 3, 6, 7a and 15 [30,31,32].Of resting position that is continually readjusted by slow these, the prime contenders for the motor that powers adaptation. Fast adaptation could theoretically participate slow adaptation are either myosin-1c [11] or myosin 7a in auditory frequency selectivity by filtering the MET [33]. Myosin-1c is concentrated in frog hair bundles at current [14] or by generating fast hair bundle movements

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that amplify the mechanical input. Hair bundles in the the protein responsible for this ‘somatic motility’ [50], and frog have the ability to mechanically amplify a it is concentrated in the lateral wall of the OHC [51]. The signal leading to spontaneous oscillations. The oscilla- voltage sensitivity of is endowed by the intracel- tions occur at low frequencies (5–50 Hz) and it has been lular binding of small anions such as Cl [52]. Targeted hypothesized that they are driven by the slow myosin deletion of prestin in mice results in an elevated threshold, motor, biasing the displacement-force relation of the hair reduced tuning, and loss of outer hair cell motility [53], bundle into a region of negative slope [43]. In contrast, which led the authors to conclude that somatic motility active bundle movements produced by Ca2þ binding to alone was responsible for the active process. This conclu- the MET channels can in principle occur at kiloHertz sion can be criticized on the grounds that any feedback frequencies, within the mammalian auditory range [17]. process involving multiple elements would be compro- Furthermore, trans-epithelial electrical stimulation of the mised if one element were eliminated. For example, isolated frog saccule experimentally evokes hair bundle abolition of the endocochlear potential (EP) by treatment oscillations at frequencies of up to 1 kHz [44]. There is with the diuretic furosemide reduces amplification about also in vivo evidence for active hair bundle motion at more 30-fold on average [54], even though the EP is not the than 1 kHz in the lizard organ [45]. However, it source of the active process. Loss of the EP will approx- remains to be seen whether or not active motion of the imately halve the MET current. With regards to the same outer hair-cell bundles can generate sufficient force to argument, halving of electromotility in prestin heterozy- produce amplification in the intact mammalian cochlea [7]. gotes [53] should have produced greater than the twofold elevation in cochlear thresholds if somatic motility were Tuning of the MET current in turtle auditory hair cells the sole source of the active process. Furthermore, al- does not require concomitant active bundle motion though OHC motility may supply the energy for amplifica- because it occurs when the bundle is displaced with a tion, there is no evidence that it is intrinsically frequency rigid stimulating probe (Figure 1; [14]). This suggests that selective. For cochlear models to produce realistically the energy associated with channel gating does not need to sharp tuning of the , the mechanical move the hair bundle in order to elicit oscillations in the feedback from the outer hair cells must occur in a fre- current. The variation in resonant frequency (58–230 Hz), quency-selective manner to ensure it supplies force at the which is within the turtle’s auditory range, may therefore appropriate phase of basilar membrane vibration [55–58]. involve differences in the MET channel. Several mechan- The required tuning has been ascribed to a mechanical isms for these differences have been proposed. The adap- resonance in the [56,58,59],butit tation rate or resonant frequency increases with a higher could equally reside in the MET channels. If the channels stereociliary Ca2þ concentration, which could be brought in mammalian OHCs operate similarly to those in turtle about either by increasing the channel’sCa2þ permeability hair cells, the transducer currents and any associated active with CF or by increasing the conductance of the MET hair bundle motion will be tuned over a frequency range channels or their number per stereocilium. The evidence imparted by variations in the speed of fast adaptation [23]. for this mechanism is that the maximum MET current increases with CF, and the rate of adaptation at a given CF Conclusions varies with the magnitude of the current [6,14]. It has been Over the past five years, evidence has emerged for at least recently shown that the channel’sCa2þ permeability does two distinct Ca2þ-mediated mechanisms of hair cell not alter with CF [46]. However, there is evidence for a transducer adaptation varying in speed, range and func- tonotopic variation in channel kinetics derived from noise tion. A fast mechanism directly affects MET channel analysis of the MET current [46], and from measure- gating, whereas a slower one regulates the mechanical ments of the time course of current activation [18].An stimulus through the action of one or more unconven- alternate view, formed on the basis of modeling hair tional myosins. Further insights into the roles of the bundle amplification, is that both fast and slow adaptation different myosins may come from the elucidation of their processes combine to produce oscillations at a frequency subcellular localization using post-embedding immuno- determined by hair bundle geometry and intracellular gold techniques, or their modification and deletion in Ca2þ dynamics [47]. transgenic animals. The fast mechanism confers tuning on the MET channels and may be an important factor in Amplification and tuning in the mammalian cochlear frequency selectivity. Because adaptation shifts cochlea the hair cell’s operating point, it may have an ancillary role In the mammalian cochlea, the outer hair cells (OHC) are in constantly adjusting and optimizing the signal-to-noise responsible for generating active mechanical amplification ratio of transduction [60]. Cloning the MET channel that provides the compressive non-linearity and frequency should illuminate how the channel interacts with Ca2þ selectivity of the basilar membrane [48]. The ability of and other subcellular components including myosins. It OHCs to elongate and shorten in response to changes in may also reveal the existence of multiple channel iso- membrane potential is thought to supply the mechanical forms with Ca2þ affinity or kinetics specialized for opera- energy for the process [49]. Prestin has been identified as tion in different frequency ranges. However, further www.current-opinion.com Current Opinion in Neurobiology 2003, 13:446–451 450 Sensory systems

electrophysiological recordings in the mammalian cochlea 15. Crawford AC, Evans MG, Fettiplace R: Activation and adaptation of transducer currents in turtle hair cells. J Physiol 1989, will be needed to settle whether or not fast adaptation is 419:405-434. present in outer hair cells and whether or not it is suf- 16. Ricci AJ, Fettiplace R: Calcium permeation of the turtle hair cell ficiently fast to participate in the active process. Evidence mechanotransducer channel and its relation to the that this may be the case was recently obtained in rat composition of endolymph. J Physiol 1998, 506:159-173. OHCs, in which transducer currents exhibited fast Ca2þ- 17. Choe Y, Magnasco M, Hudspeth AJ: A model for amplification of hair bundle motion by cyclical binding of Ca2R to dependent adaptation with a tA of less than 0.2 ms, faster mechanoelectrical transducer channels. Proc Natl Acad Sci USA than any seen in turtle auditory hair cells [61]. 1998, 95:15321-15326. 18. Fettiplace R, Crawford AC, Ricci AJ: The effects of calcium on Acknowledgements mechanotransducer channel kinetics in auditory hair cells. In Biophysics of the Cochlea: From Molecules to Models. Edited by Work in the authors’ laboratories was supported by the National Institute on Gummer AW. Singapore: World Scientific; 2003, 65-72. Deafness and Other Communicative Disorders, grants RO1 DC 01362 (to R Fettiplace) and RO1 DC 03896 (to AJ Ricci). We thank C Hackney for her 19. Howard J, Hudspeth AJ: Mechanical relaxation of the hair comments on the manuscript and N Cooper for pointing out the amplification bundle mediates adaptation in mechanoelectrical transduction discrepancy in the prestin heterozygotes. by the bullfrog’s saccular hair cell. Proc Natl Acad Sci USA 1987, 84:3064-3068. References and recommended reading 20. Walker RG, Hudspeth AJ: Calmodulin controls adaptation of Papers of particular interest, published within the annual period of mechanoelectrical transduction by hair cells of the bullfrog’s review, have been highlighted as: sacculus. Proc Natl Acad Sci USA 1996, 93:2203-2207. An  of special interest 21. Furness DN, Karkanevatos A, West B, Hackney CM:  of outstanding interest immunogold investigation of the distribution of calmodulin in the apex of the cochlear hair cells. Hear Res 2002, 173:10-20. 1. Hudspeth AJ: How the ear’s works work. Nature 1999, Myosin 1-c interacts with hair- 341:397-404. 22. Cyr JL, Dumont RA, Gillespie PG: cell receptors through its calmodulin- binding IQ domains. 2. Howard J, Hudspeth AJ: Compliance of the hair bundle J Neurosci 2002, 22:2487-2495. associated with gating of mechanoelectrical transduction 23. Ricci AJ, Crawford AC, Fettiplace R: Active hair bundle motion channels in the bullfrog’s saccular hair cell. 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Edited by at frequencies of up to 1 kHz by extracellular electrical stimulation. The Gummer AW. Singapore: World Scientific; 2003, 73-80. specificity of the response was tied to hair cell transduction by showing that it disappeared on blocking the MET channels. 61. Kennedy HJ, Evans MG, Crawford AC, Fettiplace R: Fast adaptation  of mechanoelectrical transducer channels in mammalian 45. Manley GA, Kirk DL, Ko¨ ppl C, Yates GK: In vivo evidence for a cochlear hair cells. Nat Neurosci (Published online DOI: 10.1038/  cochlear amplifier in the hair-cell bundle of lizards. Proc Natl Nn1089). Acad Sci USA 2001, 98:2826-2831. The authors use the first measurements of OHC transducer currents in Good evidence for active hair bundle motion in vivo comes from the animals after the onset of hearing to demonstrate that the MET channels properties of the otoacoustic emissions produced by electrical stimula- in mammalian cochlear hair cells possess ultra-fast activation and adap- tion of the lizard inner ear. The authors show that because the hair tation kinetics. www.current-opinion.com Current Opinion in Neurobiology 2003, 13:446–451