Prestin is required for electromotility of the outer hair cell and for the cochlear amplifier

Hearing sensitivity in mammals is enhanced by more than 40 dB (that is, 100-fold) by mechanical amplification thought to be generated by one class of cochlear sensory cells, the outer hair cells. In addition to the mechano-electrical transduction required for auditory sensation, mammalian outer hair cells also perform electromechanical transduction, whereby transmembrane voltage drives cellular length changes at audio frequencies in vitro. This electromotility is thought to arise through voltage-gated conformational changes in a membrane protein, and prestin has been proposed as this molecular motor. Here we show that targeted deletion of prestin in mice results in loss of outer hair cell electromotility in vitro and a 40-60 dB loss of cochlear sensitivity in vivo, without disruption of mechano-electrical transduction in outer hair cells. In heterozygotes, electromotility is halved and there is a twofold (about 6 dB) increase in cochlear thresholds. These results suggest that prestin is indeed the motor protein, that there is a simple and direct coupling between electromotility and cochlear amplification, and that there is no need to invoke additional active processes to explain cochlear sensitivity in the mammalian ear.     

Prestin is the motor protein of cochlear outer hair cells

The outer and inner hair cells of the mammalian cochlea perform different functions. In response to changes in membrane potential, the cylindrical outer hair cell rapidly alters its length and stiffness. These mechanical changes, driven by putative molecular motors, are assumed to produce amplification of vibrations in the cochlea that are transduced by inner hair cells. Here we have identified an abundant complementary DNA from a gene, designated Prestin, which is specifically expressed in outer hair cells. Regions of the encoded protein show moderate sequence similarity to pendrin and related sulphate/anion transport proteins. Voltage-induced shape changes can be elicited in cultured human kidney cells that express prestin. The mechanical response of outer hair cells to voltage change is accompanied by a 'gating current', which is manifested as nonlinear capacitance. We also demonstrate this nonlinear capacitance in transfected kidney cells. We conclude that prestin is the motor protein of the cochlear outer hair cell.     

Nature of the motor element in electrokinetic shape changes of cochlear outer hair cells

It is the prevailing notion that cochlear outer hair cells function as mechanical effectors as well as sensory receptors. Electrically induced changes in the shape of mammalian outer hair cells, studied in vitro, are commonly assumed to represent an aspect of their effector process that may occur in vivo. The nature of the motile process is obscure, even though none of the established cellular motors can be involved. Although it is known that the motile response is under voltage control, it is uncertain whether the stimulus is a drop in the voltage along the long axis of the cell or variation in the transmembrane potential. We have now performed experiments with cells partitioned in differing degrees between two chambers. Applied voltage stimulates the cell membrane segments in opposite polarity to an amount dependent on the partitioning. The findings show, in accordance with previous suggestions, that the driving stimulus is a local transmembrane voltage drop and that the cellular motor consists of many independent elements, distributed along the cell membrane and its associated cortical structures. We further show that the primary action of the motor elements is along the longitudinal dimension of the cell without necessarily involving changes in intracellular hydrostatic pressure. This establishes the outer hair cell motor as unique among mechanisms that control cell shape.     

Mechanoelectrical transduction of adult outer hair cells studied in a gerbil hemicochlea

Sensory receptor cells of the mammalian cochlea are morphologically and functionally dichotomized. Inner hair cells transmit auditory information to the brain, whereas outer hair cells (OHC) amplify the mechanical signal, which is then transduced by inner hair cells. Amplification by OHCs is probably mediated by their somatic motility in a mechanical feedback process. OHC motility in vivo is thought to be driven by the cell's receptor potential. The first steps towards the generation of the receptor potential are the deflection of the stereociliary bundle, and the subsequent flow of transducer current through the mechanosensitive transducer channels located at their tips. Quantitative relations between transducer currents and basilar membrane displacements are lacking, as well as their variation along the cochlear length. To address this, we simultaneously recorded OHC transducer currents (or receptor potentials) and basilar membrane motion in an excised and bisected cochlea, the hemicochlea. This preparation permits recordings from adult OHCs at various cochlear locations while the basilar membrane is mechanically stimulated. Furthermore, the stereocilia are deflected by the same means of stimulation as in vivo. Here we show that asymmetrical transducer currents and receptor potentials are significantly larger than previously thought, they possess a highly restricted dynamic range and strongly depend on cochlear location.     

Graded and nonlinear mechanical properties of sensory hairs in the mammalian hearing organ

It is generally agreed that frequency selectivity of the mammalian hearing organ is mainly due to a graded elasticity of the basilar membrane. Recent measurements of basilar membrane motion hair cell receptor potentials and neural tuning curves show that frequency selectivity can be extremely sharp. It has been suggested that in non-mammalian species there are additional tuning mechanisms in the sensory hair cells themselves, either by virtue of their electrical membrane properties or through a gradation in length of their sensory hairs. Indeed, sensory hair mechanical tuning has been demonstrated in the lizard. We have investigated the mechanical properties of sensory hair bundles in the guinea pig organ of Corti, and report here that hair-bundle stiffness increases longitudinally towards the high-frequency end of the cochlea, decreases radially towards the outer rows of cells, and is greater for excitatory than for inhibitory deflection. On the basis of these findings, we suggest that sensory hairs confer frequency-specific, nonlinear mechanical properties on the hearing organ.     

Outer hair cells in the mammalian cochlea and noise-induced hearing loss

Hair cells in the mammalian cochlea transduce mechanical stimuli into electrical signals leading to excitation of auditory nerve fibres. Because of their important role in hearing, these cells are a possible site for the loss of cochlear sensitivity that follows acoustic overstimulation. We have recorded from inner and outer hair cells (IHC, OHC) in the guinea pig cochlea during and after exposure to intense tones. Our results show functional changes in the hair cells that may explain the origin of noise-induced hearing loss. Both populations of hair cells show a reduction in amplitude and an increase in the symmetry of their acoustically evoked receptor potentials. In addition, the OHCs also suffer a sustained depolarization of the membrane potential. Significantly, the membrane and receptor potentials of the OHCs recover in parallel with cochlear sensitivity as measured by the IHC receptor potential amplitude and the auditory nerve threshold. Current theories of acoustic transduction suggest that the mechanical input to IHCs may be regulated by the OHCs. Consequently, the modified function of OHCs after acoustic overstimulation may determine the extent of the hearing loss following loud sound.     

Forces between clustered stereocilia minimize friction in the ear on a subnanometre scale

The detection of sound begins when energy derived from an acoustic stimulus deflects the hair bundles on top of hair cells. As hair bundles move, the viscous friction between stereocilia and the surrounding liquid poses a fundamental physical challenge to the ear's high sensitivity and sharp frequency selectivity. Part of the solution to this problem lies in the active process that uses energy for frequency-selective sound amplification. Here we demonstrate that a complementary part of the solution involves the fluid-structure interaction between the liquid within the hair bundle and the stereocilia. Using force measurement on a dynamically scaled model, finite-element analysis, analytical estimation of hydrodynamic forces, stochastic simulation and high-resolution interferometric measurement of hair bundles, we characterize the origin and magnitude of the forces between individual stereocilia during small hair-bundle deflections. We find that the close apposition of stereocilia effectively immobilizes the liquid between them, which reduces the drag and suppresses the relative squeezing but not the sliding mode of stereociliary motion. The obliquely oriented tip links couple the mechanotransduction channels to this least dissipative coherent mode, whereas the elastic horizontal top connectors that stabilize the structure further reduce the drag. As measured from the distortion products associated with channel gating at physiological stimulation amplitudes of tens of nanometres, the balance of viscous and elastic forces in a hair bundle permits a relative mode of motion between adjacent stereocilia that encompasses only a fraction of a nanometre. A combination of high-resolution experiments and detailed numerical modelling of fluid-structure interactions reveals the physical principles behind the basic structural features of hair bundles and shows quantitatively how these organelles are adapted to the needs of sensitive mechanotransduction.     

Stereocilin-deficient mice reveal the origin of cochlear waveform distortions

Although the cochlea is an amplifier and a remarkably sensitive and finely tuned detector of sounds, it also produces conspicuous mechanical and electrical waveform distortions. These distortions reflect nonlinear mechanical interactions within the cochlea. By allowing one tone to suppress another (masking effect), they contribute to speech intelligibility. Tones can also combine to produce sounds with frequencies not present in the acoustic stimulus. These sounds compose the otoacoustic emissions that are extensively used to screen hearing in newborns. Because both cochlear amplification and distortion originate from the outer hair cells-one of the two types of sensory receptor cells-it has been speculated that they stem from a common mechanism. Here we show that the nonlinearity underlying cochlear waveform distortions relies on the presence of stereocilin, a protein defective in a recessive form of human deafness. Stereocilin was detected in association with horizontal top connectors, lateral links that join adjacent stereocilia within the outer hair cell's hair bundle. These links were absent in stereocilin-null mutant mice, which became progressively deaf. At the onset of hearing, however, their cochlear sensitivity and frequency tuning were almost normal, although masking was much reduced and both acoustic and electrical waveform distortions were completely lacking. From this unique functional situation, we conclude that the main source of cochlear waveform distortions is a deflection-dependent hair bundle stiffness resulting from constraints imposed by the horizontal top connectors, and not from the intrinsic nonlinear behaviour of the mechanoelectrical transducer channel.     

TRPA1 is a candidate for the mechanosensitive transduction channel of vertebrate hair cells

Mechanical deflection of the sensory hair bundles of receptor cells in the inner ear causes ion channels located at the tips of the bundle to open, thereby initiating the perception of sound. Although some protein constituents of the transduction apparatus are known, the mechanically gated transduction channels have not been identified in higher vertebrates. Here, we investigate TRP (transient receptor potential) ion channels as candidates and find one, TRPA1 (also known as ANKTM1), that meets criteria for the transduction channel. The appearance of TRPA1 messenger RNA expression in hair cell epithelia coincides developmentally with the onset of mechanosensitivity. Antibodies to TRPA1 label hair bundles, especially at their tips, and tip labelling disappears when the transduction apparatus is chemically disrupted. Inhibition of TRPA1 protein expression in zebrafish and mouse inner ears inhibits receptor cell function, as assessed with electrical recording and with accumulation of a channel-permeant fluorescent dye. TRPA1 is probably a component of the transduction channel itself.     

How the ear's works work

The senses of hearing and equilibrium depend on sensory receptors called hair cells which can detect motions of atomic dimensions and respond more than 100,000 times a second. Biophysical studies suggest that mechanical forces control the opening and closing of transduction channels by acting through elastic components in each hair cell's mechanoreceptive hair bundle. Other ion channels, as well as the mechanical and hydrodynamic properties of hair bundles, tune individual hair cells to particular frequencies of stimulation.     

The role of Drosophila Piezo in mechanical nociception

Transduction of mechanical stimuli by receptor cells is essential for senses such as hearing, touch and pain. Ion channels have a role in neuronal mechanotransduction in invertebrates; however, functional conservation of these ion channels in mammalian mechanotransduction is not observed. For example, no mechanoreceptor potential C (NOMPC), a member of transient receptor potential (TRP) ion channel family, acts as a mechanotransducer in Drosophila melanogaster and Caenorhabditis elegans; however, it has no orthologues in mammals. Degenerin/epithelial sodium channel (DEG/ENaC) family members are mechanotransducers in C. elegans and potentially in D. melanogaster; however, a direct role of its mammalian homologues in sensing mechanical force has not been shown. Recently, Piezo1 (also known as Fam38a) and Piezo2 (also known as Fam38b) were identified as components of mechanically activated channels in mammals. The Piezo family are evolutionarily conserved transmembrane proteins. It is unknown whether they function in mechanical sensing in vivo and, if they do, which mechanosensory modalities they mediate. Here we study the physiological role of the single Piezo member in D. melanogaster (Dmpiezo; also known as CG8486). Dmpiezo expression in human cells induces mechanically activated currents, similar to its mammalian counterparts. Behavioural responses to noxious mechanical stimuli were severely reduced in Dmpiezo knockout larvae, whereas responses to another noxious stimulus or touch were not affected. Knocking down Dmpiezo in sensory neurons that mediate nociception and express the DEG/ENaC ion channel pickpocket (ppk) was sufficient to impair responses to noxious mechanical stimuli. Furthermore, expression of Dmpiezo in these same neurons rescued the phenotype of the constitutive Dmpiezo knockout larvae. Accordingly, electrophysiological recordings from ppk-positive neurons revealed a Dmpiezo-dependent, mechanically activated current. Finally, we found that Dmpiezo and ppk function in parallel pathways in ppk-positive cells, and that mechanical nociception is abolished in the absence of both channels. These data demonstrate the physiological relevance of the Piezo family in mechanotransduction in vivo, supporting a role of Piezo proteins in mechanosensory nociception.     

Mutations in cadherin 23 affect tip links in zebrafish sensory hair cells

Hair cells have highly organized bundles of apical projections, or stereocilia, that are deflected by sound and movement. Displacement of stereocilia stretches linkages at the tips of stereocilia that are thought to gate mechanosensory channels. To identify the molecular machinery that mediates mechanotransduction in hair cells, zebrafish mutants were identified with defects in balance and hearing. In sputnik mutants, stereociliary bundles are splayed to various degrees, with individuals displaying reduced or absent mechanotransduction. Here we show that the defects in sputnik mutants are caused by mutations in cadherin 23 (cdh23). Mutations in Cdh23 also cause deafness and vestibular defects in mice and humans, and the protein is present in hair bundles. We show that zebrafish Cdh23 protein is concentrated near the tips of hair bundles, and that tip links are absent in homozygous sputnik(tc317e) larvae. Moreover, tip links are absent in larvae carrying weak alleles of cdh23 that affect mechanotransduction but not hair bundle integrity. We conclude that Cdh23 is an essential tip link component required for hair-cell mechanotransduction.     

Sound-induced motility of isolated cochlear outer hair cells is frequency-specific

The inner ear is capable of highly selective frequency discrimination. This is achieved not only by the travelling wave of the basilar membrane in the cochlear partition, but also by the active participation of nonlinear and vulnerable elements that enhance frequency selectivity. It has been shown that isolated mammalian outer hair cells respond with a change in length when subjected to sound stimulation at a fixed frequency. Here we investigate the motile behaviour of isolated cells when the stimulus frequency is varied between 200 and 10,000 Hz. By varying the frequency and the intensity of the tone, it is possible to obtain 'tuning curves' for the motile response. We demonstrate that the cell body of solitary hair cells, free from contact with the basilar membrane, shows a sharply tuned motile behaviour. We suggest that frequency selectivity in the organ of Corti is amplified by the tuned motility of the cell body of outer hair cells.     

Deafness and renal tubular acidosis in mice lacking the K-Cl co-transporter Kcc4

Hearing depends on a high K(+) concentration bathing the apical membranes of sensory hair cells. K(+) that has entered hair cells through apical mechanosensitive channels is transported to the stria vascularis for re-secretion into the scala media(). K(+) probably exits outer hair cells by KCNQ4 K(+) channels(), and is then transported by means of a gap junction system connecting supporting Deiters' cells and fibrocytes() back to the stria vascularis. We show here that mice lacking the K(+)/Cl(-) (K-Cl) co-transporter Kcc4 (coded for by Slc12a7) are deaf because their hair cells degenerate rapidly after the beginning of hearing. In the mature organ of Corti, Kcc4 is restricted to supporting cells of outer and inner hair cells. Our data suggest that Kcc4 is important for K(+) recycling() by siphoning K(+) ions after their exit from outer hair cells into supporting Deiters' cells, where K(+) enters the gap junction pathway. Similar to some human genetic syndromes(), deafness in Kcc4-deficient mice is associated with renal tubular acidosis. It probably results from an impairment of Cl(-) recycling across the basolateral membrane of acid-secreting alpha-intercalated cells of the distal nephron.     

Ionic basis of membrane potential in outer hair cells of guinea pig cochlea

Mammalian hearing involves features not found in other species, for example, the separation of sound frequencies depends on an active control of the cochlear mechanics. The force-generating component in the cochlea is likely to be the outer hair cell (OHC), one of the two types of sensory cell through which current is gated by mechano-electrical transducer channels sited on the apical surface. Outer hair cells isolated in vitro have been shown to be motile and capable of generating forces at acoustic frequencies. The OHC membrane is not, however, electrically tuned, as found in lower vertebrates. Here we describe how the OHC resting potential is determined by a Ca2+-activated K+ conductance at the base of the cell. Two channel types with unitary sizes of 240 and 45 pS underlie this Ca2+-activated K+ conductance and we suggest that their activity is determined by a Ca2+ influx through the apical transducer channel, as demonstrated in other hair cells. This coupled system simultaneously explains the large OHC resting potentials observed in vivo and indicates how the current gated by the transducer may be maximized to generate the forces required in cochlear micromechanics.     

单畴磁铁矿磁接收器磁学模型

很多生物可以利用地磁场辨别方向和方位,而生物磁铁矿被广泛发现存在于这些生物体中,研究者认为,生物磁铁矿与周围组织(包括神经组织)构成磁接收器,它在与外界磁场的相互作用中,将外界磁场信息转化成神经信息促使生物作出相应的反应;生物磁铁矿磁接收器理论包括单畴磁铁矿磁接收器模型和超顺磁磁铁矿磁接收器模型;趋磁细菌体内存在典型的单畴磁铁矿颗粒,本文中,以趋磁细菌为例,从磁学理论角度出发,定性的探讨了单畴磁铁矿磁接收器的磁学模型和工作机制:在外磁场中,外面包围着生物膜的单畴晶体会受到力的作用,单畴晶体把力转加给生物膜,再被生物膜中的机械感受器接收到,再转变为刺激信号,引起细胞相应的运动行为;或者是,磁铁矿粒子通过与生物膜的结合,把受到的力转变为打开或关闭离子通道,影响离子的流入,跨膜电势发生改变,从而产生刺激信号;多个单畴晶体及膜的存在会导致信号的增加与放大.     

Electrokinetic shape changes of cochlear outer hair cells

Rapid mechanical changes have been associated with electrical activity in a variety of non-muscle excitable cells. Recently, mechanical changes have been reported in cochlear hair cells. Here we describe electrically evoked mechanical changes in isolated cochlear outer hair cells (OHCs) with characteristics which suggest that direct electrokinetic phenomena are implicated in the response. OHCs make up one of two mechanosensitive hair cell populations in the mammalian cochlea; their role may be to modulate the micromechanical properties of the hearing organ through mechanical feedback mechanisms. In the experiments described here, we applied sinusoidally modulated electrical potentials across isolated OHCs; this produced oscillatory elongation and shortening of the cells and oscillatory displacements of intracellular organelles. The movements were a function of the direction and strength of the electrical field, were inversely related to the ionic concentration of the medium, and occurred in the presence of metabolic uncouplers. The cylindrical shape of the OHCs and the presence of a system of membranes within the cytoplasm--laminated cisternae--may provide the anatomical substrate for electrokinetic phenomena such as electro-osmosis.     

Mechanosensitivity of mammalian auditory hair cells in vitro

Intracellular responses recorded in vitro from the cochleas of anaesthetized mammals have shown that the mechanoreceptive inner and outer hair cells are sharply tuned, accounting for many of the properties of the afferent fibres in the auditory nerve. However, in vivo it has not been possible to measure directly the excitatory mechanical input to these cells (the displacement of their mechanosensitive stereocilia) and thus to determine the relationship between the receptor potentials and displacement of their stereocilia. As a means of circumventing this technical difficulty, we have developed an organ culture of the mouse cochlea and here we describe the receptor potentials generated by the hair cells in response to direct displacement of their stereocilia.