Cadherin 23 and protocadherin 15 interact to form tip-link filaments in sensory hair cells

Hair cells of the inner ear are mechanosensors that transduce mechanical forces arising from sound waves and head movement into electrochemical signals to provide our sense of hearing and balance. Each hair cell contains at the apical surface a bundle of stereocilia. Mechanoelectrical transduction takes place close to the tips of stereocilia in proximity to extracellular tip-link filaments that connect the stereocilia and are thought to gate the mechanoelectrical transduction channel. Recent reports on the composition, properties and function of tip links are conflicting. Here we demonstrate that two cadherins that are linked to inherited forms of deafness in humans interact to form tip links. Immunohistochemical studies using rodent hair cells show that cadherin 23 (CDH23) and protocadherin 15 (PCDH15) localize to the upper and lower part of tip links, respectively. The amino termini of the two cadherins co-localize on tip-link filaments. Biochemical experiments show that CDH23 homodimers interact in trans with PCDH15 homodimers to form a filament with structural similarity to tip links. Ions that affect tip-link integrity and a mutation in PCDH15 that causes a recessive form of deafness disrupt interactions between CDH23 and PCDH15. Our studies define the molecular composition of tip links and provide a conceptual base for exploring the mechanisms of sensory impairment associated with mutations in CDH23 and PCDH15.     

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.     

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.     

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.     

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.     

Visualizing the mechanical activation of Src

The mechanical environment crucially influences many cell functions. However, it remains largely mysterious how mechanical stimuli are transmitted into biochemical signals. Src is known to regulate the integrin-cytoskeleton interaction, which is essential for the transduction of mechanical stimuli. Using fluorescent resonance energy transfer (FRET), here we develop a genetically encoded Src reporter that enables the imaging and quantification of spatio-temporal activation of Src in live cells. We introduced a local mechanical stimulation to human umbilical vein endothelial cells (HUVECs) by applying laser-tweezer traction on fibronectin-coated beads adhering to the cells. Using the Src reporter, we observed a rapid distal Src activation and a slower directional wave propagation of Src activation along the plasma membrane. This wave propagated away from the stimulation site with a speed (mean +/- s.e.m.) of 18.1 +/- 1.7 nm s(-1). This force-induced directional and long-range activation of Src was abolished by the disruption of actin filaments or microtubules. Our reporter has thus made it possible to monitor mechanotransduction in live cells with spatio-temporal characterization. We find that the transmission of mechanically induced Src activation is a dynamic process that directs signals via the cytoskeleton to spatial destinations.     

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.     

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.     

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.     

The mammalian sodium channel BNC1 is required for normal touch sensation

Of the vertebrate senses, touch is the least understood at the molecular level The ion channels that form the core of the mechanosensory complex and confer touch sensitivity remain unknown. However, the similarity of the brain sodium channel 1 (BNC1) to nematode proteins involved in mechanotransduction indicated that it might be a part of such a mechanosensor. Here we show that disrupting the mouse BNC1 gene markedly reduces the sensitivity of a specific component of mechanosensation: low-threshold rapidly adapting mechanoreceptors. In rodent hairy skin these mechanoreceptors are excited by hair movement. Consistent with this function, we found BNC1 in the lanceolate nerve endings that lie adjacent to and surround the hair follicle. Although BNC1 has been proposed to have a role in pH sensing, the acid-evoked current in cultured sensory neurons and the response of acid-stimulated nociceptors were normal in BNC1 null mice. These data identify the BNC1 channel as essential for the normal detection of light touch and indicate that BNC1 may be a central component of a mechanosensory complex.     

Force generation by mammalian hair bundles supports a role in cochlear amplification

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. The prevailing hypothesis is that this amplification stems from somatic electromotility of the outer hair cells attributable to the motor protein prestin. Thus outer hair cells contract and elongate in synchrony with the sound-evoked receptor potential. 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. Here we show that hair bundles of mammalian outer hair cells can also produce force on a submillisecond timescale linked to adaptation of the mechanotransducer 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.     

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.     

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.     

The origin of spontaneous activity in the developing auditory system

Spontaneous activity in the developing auditory system is required for neuronal survival as well as the refinement and maintenance of tonotopic maps in the brain. However, the mechanisms responsible for initiating auditory nerve firing in the absence of sound have not been determined. Here we show that supporting cells in the developing rat cochlea spontaneously release ATP, which causes nearby inner hair cells to depolarize and release glutamate, triggering discrete bursts of action potentials in primary auditory neurons. This endogenous, ATP-mediated signalling synchronizes the output of neighbouring inner hair cells, which may help refine tonotopic maps in the brain. Spontaneous ATP-dependent signalling rapidly subsides after the onset of hearing, thereby preventing this experience-independent activity from interfering with accurate encoding of sound. These data indicate that supporting cells in the organ of Corti initiate electrical activity in auditory nerves before hearing, pointing to an essential role for peripheral, non-sensory cells in the development of central auditory pathways.     

The mechanosensory protein MEC-6 is a subunit of the C. elegans touch-cell degenerin channel

Mechanosensory transduction in touch receptor neurons is believed to be mediated by DEG/ENaC (degenerin/epithelial Na+ channel) proteins in nematodes and mammals. In the nematode Caenorhabditis elegans, gain-of-function mutations in the degenerin genes mec-4 and mec-10 (denoted mec-4(d) and mec-10(d), respectively) cause degeneration of the touch cells. This phenotype is completely suppressed by mutation in a third gene, mec-6 (refs 3, 4), that is needed for touch sensitivity. This last gene is also required for the function of other degenerins. Here we show that mec-6 encodes a single-pass membrane-spanning protein with limited similarity to paraoxonases, which are implicated in human coronary heart disease. This gene is expressed in muscle cells and in many neurons, including the six touch receptor neurons. MEC-6 increases amiloride-sensitive Na+ currents produced by MEC-4(d)/MEC-10(d) by approximately 30-fold, and functions synergistically with MEC-2 (a stomatin-like protein that regulates MEC-4(d)/MEC-10(d) channel activity) to increase the currents by 200-fold. MEC-6 physically interacts with all three channel proteins. In vivo, MEC-6 co-localizes with MEC-4, and is required for punctate MEC-4 expression along touch-neuron processes. We propose that MEC-6 is a part of the degenerin channel complex that may mediate mechanotransduction in touch cells.     

Molecular basis of mechanosensory transduction

Mechanotransduction - a cell's conversion of a mechanical stimulus into an electrical signal - reveals vital features of an organism's environment. From hair cells and skin mechanoreceptors in vertebrates, to bristle receptors in flies and touch receptors in worms, mechanically sensitive cells are essential in the life of an organism. The scarcity of these cells and the uniqueness of their transduction mechanisms have conspired to slow molecular characterization of the ensembles that carry out mechanotransduction. But recent progress in both invertebrates and vertebrates is beginning to reveal the identities of proteins essential for transduction.