Mechanoelectrical Transduction Process Research Articles

Mechanosensitive Piezo channels and their potential roles in peripheral auditory perception

AbstractHearing sound and responding to external and internal mechanical stimuli requires specific proteins as mechanotransducers that convert mechanical forces into biological signals. However, our understanding of the mechanotransduction process in the inner ear is still incomplete. Mechanically activated ion channels, PIEZO1 and PIEZO2, are widely distributed throughout the body and play essential roles. Recent studies have discovered that Piezo channels are expressed in inner ear hair cells, suggesting their potential involvement in auditory perception. This review summarizes the existing discoveries about the Piezo channels, including their structure, mechanogating mechanisms, and general physiological roles, explicitly focusing on Piezo channels in the auditory systems. Piezo channels play roles in ultrasound perception, generation of anomalous current, hair cell development, and potentially in the normal mechanoelectrical transduction process of hair cells. Collectively, this review aims to provide a new perspective on the Piezo channel and its potential roles in auditory perception.

Voltage-dependent bundle motion in mammalian auditory hair cells

Auditory hair cells have bundles of protrusions on their surface held together by proteinaceous links. Sound-induced vibrations deflect these bundles, transferring force through the links to open nearby mechanically sensitive ion channels and providing our sensation of sound. An increase in bundle compliance caused by channel gating can also be detected as motion. The channels open during depolarization and the bundle moves rapidly toward shorter stereocilia, followed by a larger but slower movement toward the taller stereocilia. To determine how voltage changes affect mammalian inner hair cell (IHC) bundles, we used whole cell patch clamp and high-speed imaging to apply electrical stimulation and measure bundle motion. We found two components in the motions of freestanding IHC bundles in response to depolarization, a fast offset away from the tallest row and a slower movement back toward the tallest row. Like in turtles, the slower motion was sensitive to channel block and suggested ion flux, particularly calcium, played a role. Our findings show IHCs can rapidly respond to changes in the bundle setpoint without ion influx. This work improves our understanding of the mechano-electrical transduction process and helps address the controversial role of calcium in regulating the channel current response.

CAMP and voltage modulate rat auditory mechanotransduction by decreasing the stiffness of gating springs

Hair cells of the auditory and vestibular systems transform mechanical input into electrical potentials through the mechanoelectrical transduction process (MET). Deflection of the mechanosensory hair bundle increases tension in the gating springs that open MET channels. Regulation of MET channel sensitivity contributes to the auditory system's precision, wide dynamic range and, potentially, protection from overexcitation. Modulating the stiffness of the gating spring modulates the sensitivity of the MET process. Here, we investigated the role of cyclic adenosine monophosphate (cAMP) in rat outer hair cell MET and found that cAMP up-regulation lowers the sensitivity of the channel in a manner consistent with decreasing gating spring stiffness. Direct measurements of the mechanical properties of the hair bundle confirmed a decrease in gating spring stiffness with cAMP up-regulation. In parallel, we found that prolonged depolarization mirrored the effects of cAMP. Finally, a limited number of experiments implicate that cAMP activates the exchange protein directly activated by cAMP to mediate the changes in MET sensitivity. These results reveal that cAMP signaling modulates gating spring stiffness to affect auditory sensitivity.

Modulation of Vestibular Microphonics: A Historical Note.

Modulation of microphonics has recently been used to investigate the sensitivity of the utricle in the vestibular organ of the guinea pig. The same technique was used more than 30 years ago to obtain information on the processing of rotational stimuli in the horizontal semicircular canals of the pigeon. Data from that time were reanalysed to give a relation that describes the mechano-electrical transduction (MET) process in vestibular hair cells.

Decades-old model of slow adaptation in sensory hair cells is not supported in mammals.

Hair cells detect sound and motion through a mechano-electric transduction (MET) process mediated by tip links connecting shorter stereocilia to adjacent taller stereocilia. Adaptation is a key feature of MET that regulates a cell's dynamic range and frequency selectivity. A decades-old hypothesis proposes that slow adaptation requires myosin motors to modulate the tip-link position on taller stereocilia. This "motor model" depended on data suggesting that the receptor current decay had a time course similar to that of hair-bundle creep (a continued movement in the direction of a step-like force stimulus). Using cochlear and vestibular hair cells of mice, rats, and gerbils, we assessed how modulating adaptation affected hair-bundle creep. Our results are consistent with slow adaptation requiring myosin motors. However, the hair-bundle creep and slow adaptation were uncorrelated, challenging a critical piece of evidence upholding the motor model. Considering these data, we propose a revised model of hair cell adaptation.

The Development of Cooperative Channels Explains the Maturation of Hair Cell'S Mechanotransduction

Hearing relies on the conversion of mechanical stimuli into electrical signals. In vertebrates, this process of mechano-electrical transduction (MET) is performed by specialized receptors of the inner ear, the hair cells. Each hair cell is crowned by a hair bundle, a cluster of microvilli that pivot in response to sound vibrations, causing the opening and closing of mechanosensitive ion channels. Mechanical forces are projected onto the channels by molecular springs called tip links. Each tip link is thought to connect to a small number of MET channels that gate cooperatively and operate as a single transduction unit. Pushing the hair bundle in the excitatory direction opens the channels, after which they rapidly reclose in a process called fast adaptation. It has been experimentally observed that the hair cell's biophysical properties mature gradually during postnatal development: the maximal transduction current increases, sensitivity sharpens, transduction occurs at smaller hair-bundle displacements, and adaptation becomes faster. Similar observations have been reported during tip-link regeneration after acoustic damage. Moreover, when measured at intermediate developmental stages, the kinetics of fast adaptation varies in a given cell depending on the magnitude of the imposed displacement. The mechanisms underlying these seemingly disparate observations have so far remained elusive. Here, we show that these phenomena can all be explained by the progressive addition of MET channels of constant properties, which populate the hair bundle first as isolated entities, then progressively as clusters of more sensitive, cooperative MET channels. As the proposed mechanism relies on the difference in biophysical properties between isolated and clustered channels, this work highlights the importance of cooperative interactions between mechanosensitive ion channels for hearing.

The lhfpl5 Ohnologs lhfpl5a and lhfpl5b Are Required for Mechanotransduction in Distinct Populations of Sensory Hair Cells in Zebrafish.

Hair cells sense and transmit auditory, vestibular, and hydrodynamic information by converting mechanical stimuli into electrical signals. This process of mechano-electrical transduction (MET) requires a mechanically gated channel localized in the apical stereocilia of hair cells. In mice, lipoma HMGIC fusion partner-like 5 (LHFPL5) acts as an auxiliary subunit of the MET channel whose primary role is to correctly localize PCDH15 and TMC1 to the mechanotransduction complex. Zebrafish have two lhfpl5 genes (lhfpl5a and lhfpl5b), but their individual contributions to MET channel assembly and function have not been analyzed. Here we show that the zebrafish lhfpl5 genes are expressed in discrete populations of hair cells: lhfpl5a expression is restricted to auditory and vestibular hair cells in the inner ear, while lhfpl5b expression is specific to hair cells of the lateral line organ. Consequently, lhfpl5a mutants exhibit defects in auditory and vestibular function, while disruption of lhfpl5b affects hair cells only in the lateral line neuromasts. In contrast to previous reports in mice, localization of Tmc1 does not depend upon Lhfpl5 function in either the inner ear or lateral line organ. In both lhfpl5a and lhfpl5b mutants, GFP-tagged Tmc1 and Tmc2b proteins still localize to the stereocilia of hair cells. Using a stably integrated GFP-Lhfpl5a transgene, we show that the tip link cadherins Pcdh15a and Cdh23, along with the Myo7aa motor protein, are required for correct Lhfpl5a localization at the tips of stereocilia. Our work corroborates the evolutionarily conserved co-dependence between Lhfpl5 and Pcdh15, but also reveals novel requirements for Cdh23 and Myo7aa to correctly localize Lhfpl5a. In addition, our data suggest that targeting of Tmc1 and Tmc2b proteins to stereocilia in zebrafish hair cells occurs independently of Lhfpl5 proteins.

Nanomechanics of tip-link cadherins

Hearing and balance rely on the transduction of mechanical stimuli arising from sound waves or head movements into electrochemical signals. This archetypal mechanoelectrical transduction process occurs in the hair-cell stereocilia of the inner ear, which experience continuous oscillations driven by undulations in the endolymph in which they are immersed. The filamentous structures called tip links, formed by an intertwined thread composed of an heterotypic complex of cadherin 23 and protocadherin 15 ectodomain dimers, connect each stereocilium to the tip of the lower sterocilium, and must maintain their integrity against continuous stimulatory deflections. By using single molecule force spectroscopy, here we demonstrate that in contrast to the case of classical cadherins, tip-link cadherins are mechanoresilient structures even at the exceptionally low Ca2+ concentration of the endolymph. We also show that the D101G deafness point mutation in cadherin 23, which affects a Ca2+ coordination site, exhibits an altered mechanical phenotype at the physiological Ca2+ concentration. Our results show a remarkable case of functional adaptation of a protein’s nanomechanics to extremely low Ca2+ concentrations and pave the way to a full understanding of the mechanotransduction mechanism mediated by auditory cadherins.

The Development of Cooperative Channels Explains the Maturation of Hair Cell’s Mechanotransduction

Hearing relies on the conversion of mechanical stimuli into electrical signals. In vertebrates, this process of mechanoelectrical transduction (MET) is performed by specialized receptors of the inner ear, the hair cells. Each hair cell is crowned by a hair bundle, a cluster of microvilli that pivot in response to sound vibrations, causing the opening and closing of mechanosensitive ion channels. Mechanical forces are projected onto the channels by molecular springs called tip links. Each tip link is thought to connect to a small number of MET channels that gate cooperatively and operate as a single transduction unit. Pushing the hair bundle in the excitatory direction opens the channels, after which they rapidly reclose in a process called fast adaptation. It has been experimentally observed that the hair cell’s biophysical properties mature gradually during postnatal development: the maximal transduction current increases, sensitivity sharpens, transduction occurs at smaller hair-bundle displacements, and adaptation becomes faster. Similar observations have been reported during tip-link regeneration after acoustic damage. Moreover, when measured at intermediate developmental stages, the kinetics of fast adaptation varies in a given cell, depending on the magnitude of the imposed displacement. The mechanisms underlying these seemingly disparate observations have so far remained elusive. Here, we show that these phenomena can all be explained by the progressive addition of MET channels of constant properties, which populate the hair bundle first as isolated entities and then progressively as clusters of more sensitive, cooperative MET channels. As the proposed mechanism relies on the difference in biophysical properties between isolated and clustered channels, this work highlights the importance of cooperative interactions between mechanosensitive ion channels for hearing.

Slow oscillatory changes of DPOAE magnitude and phase after exposure to intense low-frequency sounds.

Sensitive sound detection within the mammalian cochlea is performed by hair cells surrounded by cochlear fluids. Maintenance of cochlear fluid homeostasis and tight regulation of intracellular conditions in hair cells are crucial for the auditory transduction process but can be impaired by intense sound stimulation. After a short, intense low-frequency sound, the cochlea shows the previously described "bounce phenomenon," which manifests itself as slow oscillatory changes of hearing thresholds and otoacoustic emissions. In this study, distortion product otoacoustic emissions (DPOAEs) were recorded after Mongolian gerbils were exposed to intense low-frequency sounds (200 Hz, 100 dB SPL) with different exposure times up to 1 h. After all sound exposure durations, a certain percentage of recordings (up to 80% after 1.5-min-long exposure) showed oscillatory DPOAE changes, similar to the bounce phenomenon in humans. Changes were quite uniform with respect to size and time course, and they were independent from sound exposure duration. Changes showed states of hypo- and hyperactivity with either state preceding the other. The direction of changes was suggested to depend on the static position of the cochlear operating point. As assessed with DPOAEs, no indication for a permanent damage after several or long exposure times was detected. We propose that sensitivity changes occur due to alterations of the mechanoelectrical transduction process of outer hair cells. Those alterations could be induced by different challenged homeostatic processes with slow electromotility of outer hair cells being the most plausible source of the bounce phenomenon. NEW & NOTEWORTHY Low-frequency, high-intensity sound can cause slowly cycling activity changes in the mammalian cochlea. We examined the effect of low-frequency sound duration on the degree of these alterations. We found that cochlear changes showed a stereotypical biphasic pattern independent of sound exposure duration, but the probability that significant changes occurred decreased with increasing sound duration. Despite exposure durations of up to 1 h, no permanent or transient impairments of the cochlea were detected.

Tracing Actin Filament Bundles in Three-Dimensional Electron Tomography Density Maps of Hair Cell Stereocilia.

Cryo-electron tomography (cryo-ET) is a powerful method of visualizing the three-dimensional organization of supramolecular complexes, such as the cytoskeleton, in their native cell and tissue contexts. Due to its minimal electron dose and reconstruction artifacts arising from the missing wedge during data collection, cryo-ET typically results in noisy density maps that display anisotropic XY versus Z resolution. Molecular crowding further exacerbates the challenge of automatically detecting supramolecular complexes, such as the actin bundle in hair cell stereocilia. Stereocilia are pivotal to the mechanoelectrical transduction process in inner ear sensory epithelial hair cells. Given the complexity and dense arrangement of actin bundles, traditional approaches to filament detection and tracing have failed in these cases. In this study, we introduce BundleTrac, an effective method to trace hundreds of filaments in a bundle. A comparison between BundleTrac and manually tracing the actin filaments in a stereocilium showed that BundleTrac accurately built 326 of 330 filaments (98.8%), with an overall cross-distance of 1.3 voxels for the 330 filaments. BundleTrac is an effective semi-automatic modeling approach in which a seed point is provided for each filament and the rest of the filament is computationally identified. We also demonstrate the potential of a denoising method that uses a polynomial regression to address the resolution and high-noise anisotropic environment of the density map.

Local gene therapy durably restores vestibular function in a mouse model of Usher syndrome type 1G

Our understanding of the mechanisms underlying inherited forms of inner ear deficits has considerably improved during the past 20 y, but we are still far from curative treatments. We investigated gene replacement as a strategy for restoring inner ear functions in a mouse model of Usher syndrome type 1G, characterized by congenital profound deafness and balance disorders. These mice lack the scaffold protein sans, which is involved both in the morphogenesis of the stereociliary bundle, the sensory antenna of inner ear hair cells, and in the mechanoelectrical transduction process. We show that a single delivery of the sans cDNA by the adenoassociated virus 8 to the inner ear of newborn mutant mice reestablishes the expression and targeting of the protein to the tips of stereocilia. The therapeutic gene restores the architecture and mechanosensitivity of stereociliary bundles, improves hearing thresholds, and durably rescues these mice from the balance defects. Our results open up new perspectives for efficient gene therapy of cochlear and vestibular disorders by showing that even severe dysmorphogenesis of stereociliary bundles can be corrected.

Genetics of auditory mechano-electrical transduction

The hair bundles of cochlear hair cells play a central role in the auditory mechano-electrical transduction (MET) process. The identification of MET components and of associated molecular complexes by biochemical approaches is impeded by the very small number of hair cells within the cochlea. In contrast, human and mouse genetics have proven to be particularly powerful. The study of inherited forms of deafness led to the discovery of several essential proteins of the MET machinery, which are currently used as entry points to decipher the associated molecular networks. Notably, MET relies not only on the MET machinery but also on several elements ensuring the proper sound-induced oscillation of the hair bundle or the ionic environment necessary to drive the MET current. Here, we review the most significant advances in the molecular bases of the MET process that emerged from the genetics of hearing.

Mutations of the Mouse ELMO Domain Containing 1 Gene (Elmod1) Link Small GTPase Signaling to Actin Cytoskeleton Dynamics in Hair Cell Stereocilia

Stereocilia, the modified microvilli projecting from the apical surfaces of the sensory hair cells of the inner ear, are essential to the mechanoelectrical transduction process underlying hearing and balance. The actin-filled stereocilia on each hair cell are tethered together by fibrous links to form a highly patterned hair bundle. Although many structural components of hair bundles have been identified, little is known about the signaling mechanisms that regulate their development, morphology, and maintenance. Here, we describe two naturally occurring, allelic mutations that result in hearing and balance deficits in mice, named roundabout (rda) and roundabout-2J (rda2J). Positional cloning identified both as mutations of the mouse ELMO domain containing 1 gene (Elmod1), a poorly characterized gene with no previously reported mutant phenotypes. The rda mutation is a 138 kb deletion that includes exons 1–5 of Elmod1, and rda2J is an intragenic duplication of exons 3–8 of Elmod1. The deafness associated with these mutations is caused by cochlear hair cell dysfunction, as indicated by conspicuous elongations and fusions of inner hair cell stereocilia and progressive degeneration of outer hair cell stereocilia. Mammalian ELMO-family proteins are known to be involved in complexes that activate small GTPases to regulate the actin cytoskeleton during phagocytosis and cell migration. ELMOD1 and ELMOD2 recently were shown to function as GTPase-activating proteins (GAPs) for the Arf family of small G proteins. Our finding connecting ELMOD1 deficiencies with stereocilia dysmorphologies thus establishes a link between the Ras superfamily of small regulatory GTPases and the actin cytoskeleton dynamics of hair cell stereocilia.

Mutations in PMCA2 and hereditary deafness: A molecular analysis of the pump defect

The inner ear converts sound waves into hearing signals through the mechanoelectrical transduction (MET) process. Deflection of the stereocilia bundle of hair cells causes the opening of channels that allow the entry of endolymph K + and Ca 2+. Ca 2+ that enters is crucial to the hearing process and is exported to the endolymph by the plasma membrane Ca 2+ pump (isoform PMCA2w/a): disturbances of the balance between Ca 2+ penetration and ejection, e.g. by pump mutations, generate deafness. Hearing loss caused by PMCA defects is frequently exacerbated by mutations in cadherin 23, a single pass stereociliar Ca 2+ binding protein that forms the tip links which permit the deflection of the stereocilia bundle and thus the opening of the MET channels. The PMCA2w/a pump ejects Ca 2+ to the endolymph even in the absence of the natural activator calmodulin. This satisfies the special Ca 2+ homeostasis requirements of the stereocilia/endolymph system. Here we have analyzed a mice and a human previously described pump mutant. The human mutant only exacerbated the deafness produced by a cadherin 23 mutation. The murine mutant overexpressed in model cells displayed an evident defect both in the basal activity of the pump and in the long range ejection of Ca 2+, the human mutant instead failed to impair the Ca 2+ ejection by the pump.

The plasma membrane calcium pump in the hearing process: physiology and pathology

Mammalian cells express four different plasma membrane Ca(2+) ATPases. Two of them (PMCA1 and PMCA4) are expressed ubiquitously, and are considered housekeeping isoforms. Two (PMCA2 and PMCA4) have tissue restricted distribution. They are abundantly expressed in the brain and in nervous tissue-derived cell types. The primary transcripts of all PMCAs undergo alternative splicing, generating a large number of additional isoforms. Splicing occurs at site A, in the N-terminal moiety of the pump, and at site C, within the C-terminal calmodulin binding domain: The pumps are canonical targets of calmodulin stimulation. The site C insertion leads to a truncation of the pump about 50 residues short of the original C-terminal. One of the pumps (PMCA2) has special properties: It displays high activity even in the absence of the natural activator calmodulin, and has a particularly complex pattern of alternative splicing at both sites A and C. A variant of the PMCA2 pump containing an insert at site A and truncated C-terminally is the resident isoform of the pump in the stereocilia of hair cells of the inner ear. It exports Ca(2+) to the endolymph that bathes the stereocilia less efficiently than the full length, non-inserted PMCA2 pump. The proper functioning of hair cells demands the precise maintenance of the Ca(2+) balance between hair cells and the endolymph. Disturbances in the balance affect the process of mechano-electrical transduction, which depends on the ability of the stereociliar bundle to deflect in response to sound waves. The tip links that organize the bundle are formed by the Ca(2+) binding protein cadherin 23 and by protocadherin 15: Disturbances of the Ca(2+) binding by cadherin 23 and/or of the ability of the PMCA2 variant of the stereocilia to export Ca(2+) to the endolymph generate hearing loss phenotypes. Such phenotypes have now been described in mice and humans. In some cases they are linked to mutations of both cadherin 23 and the PMCA2 pump, but in other cases they may be generated by mutations of particular severity in only one of the two proteins. The PMCA2 defect that leads to deafness has now been analyzed molecularly: It affects the long range, unstimulated ability of PMCA2 to export Ca(2+).

Hair cell inspired mechanotransduction with a gel-supported, artificial lipid membrane

A gel-supported lipid bilayer formed at the base of an artificial hair is used as the transduction element in an artificial, membrane-based hair cell sensor inspired by the structure and function of mammalian hair cells. This paper describes the initial fabrication and characterization of a bioderived, soft-material alternative to previous artificial hair cells that used the transduction properties of synthetic materials for flow and touch sensing. Under an applied air flow, the artificial hair structure vibrates, triggering a picoamp-level electrical current across the lipid bilayer. Experimental analysis of this mechanoelectrical transduction process supports the hypothesis that the current is produced by a time-varying change in the capacitance of the membrane caused by the vibration of the hair. Specifically, frequency analysis of both the motion of the hair and the measured current show that both phenomena occur at similar frequencies (0.1–1.0 kHz), which suggests that changes in capacitance occur as a result of membrane bending during excitation. In this paper, the bilayer-based hair cell sensor is experimentally characterized to understand the effects of transmembrane potential, the applied air flow, and the dimensions of the hair.

Amplifying effect of a release mechanism for fast adaptation in the hair bundle

A "release" mechanism, which has been experimentally observed as the fast component in the hair bundle's response to mechanical stimulation, appears similar to common mechanical relaxation with a damping effect. This observation is puzzling because such a response is expected to have an amplifying role in the mechanoelectrical transduction process in hair cells. Here it is shown that a release mechanism can indeed have a role in amplification, if it is associated with negative stiffness due to the gating of the mechonoelectric transducer channel.

Effects of acoustic trauma on acoustic enhancement of electrically evoked otoacoustic emissions

Moderate acoustic trauma results in decreased cochlear sensitivity and frequency selectivity. This decrease is believed to be caused by damage to the cochlear amplifier that is associated with outer hair cells (OHCs) and their nonlinear electromechanical characteristics. A consequence of OHC nonlinearity is the acoustic enhancement effect, in which low-frequency electrically evoked otoacoustic emissions are enhanced by a simultaneous tone. The present study found that acoustic trauma reduced the acoustic enhancement effect and this reduction is correlated with the N1 threshold at the electrode site. This result is consistent with the theory that trauma affects the mechanoelectric transduction process, thus affecting cochlear mechanical nonlinearity. Acoustic trauma also reduced the cochlear microphonic in a way that suggests that the number of functioning tension-gated channels and the stiffness of the gating springs were decreased. In some cases, the electromechanical transduction process was also found to be affected by acoustic trauma.

Distribution of sensory receptors in joints of the upper cervical column in the laboratory marsupial Monodelphis domestica

In order to investigate the sensory innervation, the upper cervical spine of a small laboratory marsupial (monodelphis domestica) was examined with serial section light microscopy and re-embedding of selected sections for electron microscopy. Large numbers of free nerve endings supplied by A delta- and C-fibres were found in the longitudinal ligaments and facet joint capsules. Electron microscopically, areas of direct contact between axon and collagen fibres of the surrounding connective tissue separated only by the basal lamina were observed. Such structural adaptations suggest mechanoreceptive or polymodal nociceptive functions. In addition, about 100 small lamellated corpuscles were found in the longitudinal ligaments mainly concentrated around the first intervertebral disk. Electron microscopy shows finger-like processes extending from the axon terminal into the inner core lamellae. These are the likely sites of the mechanoelectric transduction process. Smaller numbers of lamellated corpuscles were seen in the lower intervertebral disks and facet joint capsules. Lamellated corpuscles are known to function as rapidly adapting mechanoreceptors supplementing information supplied by muscle spindles to the CNS about position and movement of the cervical spine.