Hair Cell Mechanotransducer Channel Research Articles

TMC7 deficiency causes acrosome biogenesis defects and male infertility in mice.

Transmembrane channel-like (TMC) proteins are a highly conserved ion channel family consisting of eight members (TMC1-TMC8) in mammals. TMC1/2 are components of the mechanotransduction channel in hair cells, and mutations of TMC1/2 cause deafness in humans and mice. However, the physiological roles of other TMC proteins remain largely unknown. Here, we show that Tmc7 is specifically expressed in the testis and that it is required for acrosome biogenesis during spermatogenesis. Tmc7-/- mice exhibited abnormal sperm head, disorganized mitochondrial sheaths, and reduced number of elongating spermatids, similar to human oligo-astheno-teratozoospermia. We further demonstrate that TMC7 is colocalized with GM130 at the cis-Golgi region in round spermatids. TMC7 deficiency leads to aberrant Golgi morphology and impaired fusion of Golgi-derived vesicles to the developing acrosome. Moreover, upon loss of TMC7 intracellular ion homeostasis is impaired and ROS levels are increased, which in turn causes Golgi and endoplasmic reticulum stress. Taken together, these results suggest that TMC7 is required to maintain pH and ion homeostasis, which is needed for acrosome biogenesis. Our findings unveil a novel role for TMC7 in acrosome biogenesis during spermiogenesis.

LHFPL5 is a key element in force transmission from the tip link to the hair cell mechanotransducer channel.

During auditory transduction, sound-evoked vibrations of the hair cell stereociliary bundles open mechanotransducer (MET) ion channels via tip links extending from one stereocilium to its neighbor. How tension in the tip link is delivered to the channel is not fully understood. The MET channel comprises a pore-forming subunit, transmembrane channel-like protein (TMC1 or TMC2), aided by several accessory proteins, including LHFPL5 (lipoma HMGIC fusion partner-like 5). We investigated the role of LHFPL5 in transduction by comparing MET channel activation in outer hair cells of Lhfpl5-/- knockout mice with those in Lhfpl5+/- heterozygotes. The 10 to 90 percent working range of transduction in Tmc1+/+; Lhfpl5+/- was 52 nm, from which the single-channel gating force, Z, was evaluated as 0.34 pN. However, in Tmc1+/+; Lhfpl5-/- mice, the working range increased to 123 nm and Z more than halved to 0.13 pN, indicating reduced sensitivity. Tip link tension is thought to activate the channel via a gating spring, whose stiffness is inferred from the stiffness change on tip link destruction. The gating stiffness was ~40 percent of the total bundle stiffness in wild type but was virtually abolished in Lhfpl5-/-, implicating LHFPL5 as a principal component of the gating spring. The mutation Tmc1 p.D569N reduced the LHFPL5 immunolabeling in the stereocilia and like Lhfpl5-/- doubled the MET working range, but other deafness mutations had no effect on the dynamic range. We conclude that tip-link tension is transmitted to the channel primarily via LHFPL5; residual activation without LHFPL5 may occur by direct interaction between PCDH15 and TMC1.

Mutation-agnostic RNA interference with engineered replacement rescues Tmc1-related hearing loss.

Hearing loss is the most common sensory deficit, of which genetic etiologies are a frequent cause. Dominant and recessive mutations in TMC1, a gene encoding the pore-forming subunit of the hair cell mechanotransduction channel, cause DFNA36 and DFNB7/11, respectively, accounting for ∼2% of genetic hearing loss. Previous work has established the efficacy of mutation-targeted RNAi in treatment of murine models of autosomal dominant non-syndromic deafness. However, application of such approaches is limited by the infeasibility of development and validation of novel constructs for each variant. We developed an allele-non-specific approach consisting of mutation-agnostic RNAi suppression of both mutant and WT alleles, co-delivered with a knockdown-resistant engineered WT allele with or without the use of woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) to augment transgene expression. This therapeutic construct was delivered into the mature murine model of DFNA36 with an AAV vector and achieved robust hair cell and auditory brainstem response preservation. However, WPRE-enhanced Tmc1 expression resulted in inferior outcomes, suggesting a role for gene dosage optimization in future TMC1 gene therapy development.

Prolonged Dexamethasone Exposure Enhances Zebrafish Lateral-Line Regeneration But Disrupts Mitochondrial Homeostasis and Hair Cell Function

The synthetic glucocorticoid dexamethasone is commonly used to treat inner ear disorders. Previous work in larval zebrafish has shown that dexamethasone treatment enhances hair cell regeneration, yet dexamethasone has also been shown to inhibit regeneration of peripheral nerves after lesion. We therefore used the zebrafish model to determine the impact of dexamethasone treatment on lateral-line hair cells and primary afferents. To explore dexamethasone in the context of regeneration, we used copper sulfate (CuSO4) to induce hair cell loss and retraction of nerve terminals, and then allowed animals to recover in dexamethasone for 48 h. Consistent with previous work, we observed significantly more regenerated hair cells in dexamethasone-treated larvae. Importantly, we found that the afferent processes beneath neuromasts also regenerated in the presence of dexamethasone and formed an appropriate number of synapses, indicating that innervation of hair cells was not inhibited by dexamethasone. In addition to regeneration, we also explored the effects of prolonged dexamethasone exposure on lateral-line homeostasis and function. Following dexamethasone treatment, we observed hyperpolarized mitochondrial membrane potentials (ΔΨm) in neuromast hair cells and supporting cells. Hair cells exposed to dexamethasone were also more vulnerable to neomycin-induced cell death. In response to a fluid-jet delivered saturating stimulus, calcium influx through hair cell mechanotransduction channels was significantly reduced, yet presynaptic calcium influx was unchanged. Cumulatively, these observations indicate that dexamethasone enhances hair cell regeneration in lateral-line neuromasts, yet also disrupts mitochondrial homeostasis, making hair cells more vulnerable to ototoxic insults and possibly impacting hair cell function.

The speed of the hair cell mechanotransducer channel revealed by fluctuation analysis.

Although mechanoelectrical transducer (MET) channels have been extensively studied, uncertainty persists about their molecular architecture and single-channel conductance. We made electrical measurements from mouse cochlear outer hair cells (OHCs) to reexamine the MET channel conductance comparing two different methods. Analysis of fluctuations in the macroscopic currents showed that the channel conductance in apical OHCs determined from nonstationary noise analysis was about half that of single-channel events recorded after tip link destruction. We hypothesized that this difference reflects a bandwidth limitation in the noise analysis, which we tested by simulations of stochastic fluctuations in modeled channels. Modeling indicated that the unitary conductance depended on the relative values of the channel activation time constant and the applied low-pass filter frequency. The modeling enabled the activation time constant of the channel to be estimated for the first time, yielding a value of only a few microseconds. We found that the channel conductance, assayed with both noise and recording of single-channel events, was reduced by a third in a new deafness mutant, Tmc1 p.D528N. Our results indicate that noise analysis is likely to underestimate MET channel amplitude, which is better characterized from recordings of single-channel events.

PIEZO2 mediates ultrasonic hearing via cochlear outer hair cells in mice

Ultrasonic hearing and vocalization are the physiological mechanisms controlling echolocation used in hunting and navigation by microbats and bottleneck dolphins and for social communication by mice and rats. The molecular and cellular basis for ultrasonic hearing is as yet unknown. Here, we show that knockout of the mechanosensitive ion channel PIEZO2 in cochlea disrupts ultrasonic- but not low-frequency hearing in mice, as shown by audiometry and acoustically associative freezing behavior. Deletion of Piezo2 in outer hair cells (OHCs) specifically abolishes associative learning in mice during hearing exposure at ultrasonic frequencies. Ex vivo cochlear Ca2+ imaging has revealed that ultrasonic transduction requires both PIEZO2 and the hair-cell mechanotransduction channel. The present study demonstrates that OHCs serve as effector cells, combining with PIEZO2 as an essential molecule for ultrasonic hearing in mice.

Dissociating antibacterial from ototoxic effects of gentamicin C-subtypes

Gentamicin is a potent broad-spectrum aminoglycoside antibiotic whose use is hampered by ototoxic side-effects. Hospital gentamicin is a mixture of five gentamicin C-subtypes and several impurities of various ranges of nonexact concentrations. We developed a purification strategy enabling assaying of individual C-subtypes and impurities for ototoxicity and antimicrobial activity. We found that C-subtypes displayed broad and potent in vitro antimicrobial activities comparable to the hospital gentamicin mixture. In contrast, they showed different degrees of ototoxicity in cochlear explants, with gentamicin C2b being the least and gentamicin C2 the most ototoxic. Structure-activity relationships identified sites in the C4'-C6' region on ring I that reduced ototoxicity while preserving antimicrobial activity, thus identifying targets for future drug design and mechanisms for hair cell toxicity. Structure-activity relationship data suggested and electrophysiological data showed that the C-subtypes both bind and permeate the hair cell mechanotransducer channel, with the stronger the binding the less ototoxic the compound. Finally, both individual and reformulated mixtures of C-subtypes demonstrated decreased ototoxicity while maintaining antimicrobial activity, thereby serving as a proof-of-concept of drug reformulation to minimizing ototoxicity of gentamicin in patients.

Molecular Crux of Hair Cell Mechanotransduction Machinery

Although 62 years have elapsed since the first report of hereditary deafness in a mouse strain, the molecular mechanism of hair cell mechanotransduction remains elusive. Three recent studies present crucial insights into the molecular crux of hair cell mechanotransduction machinery.

Ankyrin Is An Intracellular Tether for TMC Mechanotransduction Channels

SummaryMechanotransduction channels have been proposed as force sensors in various physiological processes, such as hearing and touch. In particular, TMC1 has been shown to constitute the pore of hair cell mechanotransduction channels, but little is known about how force is sensed by TMC channels. Here, we identify UNC-44/ankyrin as an essential component of the TMC-1 mechanotransduction channel complex in the sensory cilia of Caenorhabditis elegans mechanoreceptor neurons. Ankyrin binds indirectly to TMC-1 via evolutionarily conserved CIB proteins, which are required for TMC-1-mediated mechanosensation in C. elegans OLQ neurons and body wall muscles. Mechanosensory activity conferred by ectopically expressed TMCs in mechanoinsensitive neurons depends on both ankyrin and CIB proteins, indicating that the ankyrin-CIB subcomplex is required for TMC mechanosensitivity. Our work indicates that ankyrin is a long-sought intracellular tether that transmits force to TMC mechanotransduction channels.

Direction Selectivity in Drosophila Proprioceptors Requires the Mechanosensory Channel Tmc

Drosophila Transmembrane channel-like (Tmc) is a protein that functions in larval proprioception. The closely related TMC1 protein is required for mammalian hearing and is a pore-forming subunit of the hair cell mechanotransduction channel. In hair cells, TMC1 is gated by small deflections of microvilli thatproduce tension on extracellular tip-links that connect adjacent villi. How Tmc might be gated in larval proprioceptors, which are neurons having a morphology that is completely distinct from hair cells, is unknown. Here, we have used high-speed confocal microscopy both to measure displacements of proprioceptive sensory dendrites during larval movement and to optically measure neural activity of the moving proprioceptors. Unexpectedly, the pattern of dendrite deformation for distinct neurons was unique and differed depending on the direction of locomotion: ddaE neuron dendrites were strongly curved by forward locomotion, while the dendrites of ddaD were more strongly deformed by backward locomotion. Furthermore, GCaMP6f calcium signals recorded in the proprioceptive neurons during locomotion indicated tuning to the direction of movement. ddaE showed strong activation during forward locomotion, while ddaD showed responses that were strongest during backward locomotion. Peripheral proprioceptive neurons in animals mutant for Tmc showed a near-complete loss of movement related calcium signals. As the strength of the responses of wild-type animals was correlated with dendrite curvature, we propose that Tmc channels may be activated by membrane curvature in dendrites that are exposed to strain. Our findings begin to explain how distinct cellular systems rely on a common molecular pathway for mechanosensory responses.

Structural Relationship between the Putative Hair Cell Mechanotransduction Channel TMC1 and TMEM16 Proteins

Our senses of touch, hearing, balance, and proprioception depend on mechanically gated ion channels, which transduce mechanical energy into electrical signals that are transmitted to the brain. Previous studies on the mechanisms of hearing have elucidated the biophysical properties of the mechanotransduction (MET) channel essential for hearing, yet its molecular identity and structure remain elusive. The trantasmembrane channel-like 1 protein (TMC1) localizes to the site of the MET channel, interacts with the tip-link responsible for mechanical gating, and genetic alterations in TMC1 alter MET channel properties and cause deafness, supporting the hypothesis that TMC1 forms the MET channel. We generated a model of TMC1 based on X-ray and cryo-EM structures of TMEM16 proteins, revealing the presence of a large cavity near the protein-lipid interface that also harbors two TMC1 mutations (hTMC1 p.M418K and p.D572N/H) that cause autosomal dominant hearing loss, suggesting that it could function as a permeation pathway. We also find that hair cells are permeable to 3 kDa dextran labeled with Texas Red, and that dextran permeation requires TMC1/2 proteins and functional MET channels, supporting the presence of a large permeation pathway and the hypothesis that TMC1 is a pore forming subunit of the MET channel complex.

Structural relationship between the putative hair cell mechanotransduction channel TMC1 and TMEM16 proteins.

The hair cell mechanotransduction (MET) channel complex is essential for hearing, yet it's molecular identity and structure remain elusive. The transmembrane channel-like 1 (TMC1) protein localizes to the site of the MET channel, interacts with the tip-link responsible for mechanical gating, and genetic alterations in TMC1 alter MET channel properties and cause deafness, supporting the hypothesis that TMC1 forms the MET channel. We generated a model of TMC1 based on X-ray and cryo-EM structures of TMEM16 proteins, revealing the presence of a large cavity near the protein-lipid interface that also harbors the Beethoven mutation, suggesting that it could function as a permeation pathway. We also find that hair cells are permeable to 3 kDa dextrans, and that dextran permeation requires TMC1/2 proteins and functional MET channels, supporting the presence of a large permeation pathway and the hypothesis that TMC1 is a pore forming subunit of the MET channel complex.

The murine catecholamine methyltransferase mTOMT is essential for mechanotransduction by cochlear hair cells.

Hair cells of the cochlea are mechanosensors for the perception of sound. Mutations in the LRTOMT gene, which encodes a protein with homology to the catecholamine methyltransferase COMT that is linked to schizophrenia, cause deafness. Here, we show that Tomt/Comt2, the murine ortholog of LRTOMT, has an unexpected function in the regulation of mechanotransduction by hair cells. The role of mTOMT in hair cells is independent of mTOMT methyltransferase function and mCOMT cannot substitute for mTOMT function. Instead, mTOMT binds to putative components of the mechanotransduction channel in hair cells and is essential for the transport of some of these components into the mechanically sensitive stereocilia of hair cells. Our studies thus suggest functional diversification between mCOMT and mTOMT, where mTOMT is critical for the assembly of the mechanotransduction machinery of hair cells. Defects in this process are likely mechanistically linked to deafness caused by mutations in LRTOMT/Tomt.

Molecular Identity of the Mechanotransduction Channel in Hair Cells: Not Quiet There Yet.

Hair cells in the mammalian cochlea are specialized mechanosensory cells that convert sound-induced vibrations into electrochemical signals. The molecular composition of the mechanotransduction channel underlying auditory perception has been difficult to define. The study of genes that are linked to inherited forms of deafness has recently provided tantalizing clues. Current findings indicate that the mechanotransduction channel in hair cells is a complex molecular machine. Four different proteins (TMHS/LHFPL5, TMIE, TMC1, and TMC2) have so far been linked to the transduction channel, but which proteins contribute to the channel pore still needs to be determined. Current evidence also suggests that the channel complex may contain additional, yet to be identified components.

Adaptation Independent Modulation of Auditory Hair Cell Mechanotransduction Channel Open Probability Implicates a Role for the Lipid Bilayer.

The auditory system is able to detect movement down to atomic dimensions. This sensitivity comes in part from mechanisms associated with gating of hair cell mechanoelectric transduction (MET) channels. MET channels, located at the tops of stereocilia, are poised to detect tension induced by hair bundle deflection. Hair bundle deflection generates a force by pulling on tip-link proteins connecting adjacent stereocilia. The resting open probability (P(open)) of MET channels determines the linearity and sensitivity to mechanical stimulation. Classically, P(open) is regulated by a calcium-sensitive adaptation mechanism in which lowering extracellular calcium or depolarization increases P(open). Recent data demonstrated that the fast component of adaptation is independent of both calcium and voltage, thus requiring an alternative explanation for the sensitivity of P(open) to calcium and voltage. Using rat auditory hair cells, we characterize a mechanism, separate from fast adaptation, whereby divalent ions interacting with the local lipid environment modulate resting P(open). The specificity of this effect for different divalent ions suggests binding sites that are not an EF-hand or calmodulin model. GsMTx4, a lipid-mediated modifier of cationic stretch-activated channels, eliminated the voltage and divalent sensitivity with minimal effects on adaptation. We hypothesize that the dual mechanisms (lipid modulation and adaptation) extend the dynamic range of the system while maintaining adaptation kinetics at their maximal rates.

Designer aminoglycosides prevent cochlear hair cell loss and hearing loss.

Bacterial infections represent a rapidly growing challenge to human health. Aminoglycosides are widely used broad-spectrum antibiotics, but they inflict permanent hearing loss in up to ~50% of patients by causing selective sensory hair cell loss. Here, we hypothesized that reducing aminoglycoside entry into hair cells via mechanotransducer channels would reduce ototoxicity, and therefore we synthesized 9 aminoglycosides with modifications based on biophysical properties of the hair cell mechanotransducer channel and interactions between aminoglycosides and the bacterial ribosome. Compared with the parent aminoglycoside sisomicin, all 9 derivatives displayed no or reduced ototoxicity, with the lead compound N1MS 17 times less ototoxic and with reduced penetration of hair cell mechanotransducer channels in rat cochlear cultures. Both N1MS and sisomicin suppressed growth of E. coli and K. pneumoniae, with N1MS exhibiting superior activity against extended spectrum β lactamase producers, despite diminished activity against P. aeruginosa and S. aureus. Moreover, systemic sisomicin treatment of mice resulted in 75% to 85% hair cell loss and profound hearing loss, whereas N1MS treatment preserved both hair cells and hearing. Finally, in mice with E. coli-infected bladders, systemic N1MS treatment eliminated bacteria from urinary tract tissues and serially collected urine samples, without compromising auditory and kidney functions. Together, our findings establish N1MS as a nonototoxic aminoglycoside and support targeted modification as a promising approach to generating nonototoxic antibiotics.

Modulation of Rat Auditory Hair Cell Mechanotransduction Channel Resting Open Probability Implicates a Role for the Lipid Bilayer

The auditory system is the most sensitive mechanosensory system known, able to detect movement down to atomic dimensions (Bialek. 1987. Annu. Rev. Biophys. Biophys. Chem. 16: 455-478). This sensitivity is at least partly attributed to the mechanisms associated with gating of hair cell mechanotransduction (MET) channels, where at rest approximately 50% of the channels are open, positioning the channels at the steepest part of their activation curve (Johnson et al. 2011. Neuron 70, 1143-1154). Previously, a calcium-regulated adaptation process was thought to control the resting open probability of MET channels (Farris, Wells, and Ricci. 2006. J. Neurosci. 26: 12526-12536). However, mammalian cochlear hair cell adaptation is not driven by calcium and low external calcium effects on resting open probability occur via an uncharacterized extracellular mechanism (Peng, Effertz, and Ricci. 2013. Neuron 80: 960-972). Upon further characterization of the extracellular mechanism, we find that large radii divalent ions act similarly to calcium intimating a nonspecific divalent ion mechanism. We show that hair cell depolarization also increases the resting open probability, and acts through a similar mechanism as the external calcium. Finally, we find that GsMTx4, a purported lipid-mediated stretch-activated channel modifier, blocks both the voltage and the low external calcium effects on resting open probability with only a minor decrease in adaptation. These results suggest a new, probably lipid-mediated, mechanism of calcium modulation of the hair cell MET channel independent of adaptation. Additionally, these results suggest that auditory mechanotransduction channel may have similar mechanisms to other mechanically-sensitive ion channels.This work was supported by RO1 DC0003896 to AJR, F32 DC010975 and K99 DC013299 to AWP, and NIDCD Core Grant P30-44992.

Subunit determination of the conductance of hair-cell mechanotransducer channels

Cochlear hair cells convert sound stimuli into electrical signals by gating of mechanically sensitive ion channels in their stereociliary (hair) bundle. The molecular identity of this ion channel is still unclear, but its properties are modulated by accessory proteins. Two such proteins are transmembrane channel-like protein isoform 1 (TMC1) and tetraspan membrane protein of hair cell stereocilia (TMHS, also known as lipoma HMGIC fusion partner-like 5, LHFPL5), both thought to be integral components of the mechanotransduction machinery. Here we show that, in mice harboring an Lhfpl5 null mutation, the unitary conductance of outer hair cell mechanotransducer (MT) channels was reduced relative to wild type, and the tonotopic gradient in conductance, where channels from the cochlear base are nearly twice as conducting as those at the apex, was almost absent. The macroscopic MT current in these mutants was attenuated and the tonotopic gradient in amplitude was also lost, although the current was not completely extinguished. The consequences of Lhfpl5 mutation mirror those due to Tmc1 mutation, suggesting a part of the MT-channel conferring a large and tonotopically variable conductance is similarly disrupted in the absence of Lhfpl5 or Tmc1. Immunolabelling demonstrated TMC1 throughout the stereociliary bundles in wild type but not in Lhfpl5 mutants, implying the channel effect of Lhfpl5 mutations stems from down-regulation of TMC1. Both LHFPL5 and TMC1 were shown to interact with protocadherin-15, a component of the tip link, which applies force to the MT channel. We propose that titration of the TMC1 content of the MT channel sets the gradient in unitary conductance along the cochlea.

TMIE Is an Essential Component of the Mechanotransduction Machinery of Cochlear Hair Cells

Hair cells are the mechanosensory cells of the inner ear. Mechanotransduction channels in hair cells are gated by tip links. The molecules that connect tip links to transduction channels are not known. Here we show that the transmembrane protein TMIE forms a ternary complex with the tip-link component PCDH15 and its binding partner TMHS/LHFPL5. Alternative splicing of the PCDH15 cytoplasmic domain regulates formation of this ternary complex. Transducer currents are abolished by a homozygous Tmie-null mutation, and subtle Tmie mutations that disrupt interactions between TMIE and tip links affect transduction, suggesting that TMIE is an essential component of the hair cell's mechanotransduction machinery that functionally couples the tip link to the transduction channel. The multisubunit composition of the transduction complex and the regulation of complex assembly by alternative splicing is likely critical for regulating channel properties in different hair cells and along the cochlea's tonotopic axis.

Zonal variations in K+ currents in vestibular crista calyx terminals.

We developed a rodent crista slice to investigate regional variations in electrophysiological properties of vestibular afferent terminals. Thin transverse slices of the gerbil crista ampullaris were made and electrical properties of calyx terminals in central zones (CZ) and peripheral zones (PZ) compared with whole cell patch clamp. Spontaneous action potential firing was observed in 25% of current-clamp recordings and was either regular or irregular in both zones. Firing was abolished when extracellular choline replaced Na(+) but persisted when hair cell mechanotransduction channels or calyx AMPA receptors were blocked. This suggests that ion channels intrinsic to the calyx can generate spontaneous firing. In response to depolarizing voltage steps, outward K(+) currents were observed at potentials above -60 mV. K(+) currents in PZ calyces showed significantly more inactivation than currents in CZ calyces. Underlying K(+) channel populations contributing to these differences were investigated. The KCNQ channel blocker XE991 dihydrochloride blocked a slowly activating, sustained outward current in both PZ and CZ calyces, indicating the presence of KCNQ channels. Mean reduction was greatest in PZ calyces. XE991 also reduced action potential firing frequency in CZ and PZ calyces and broadened mean action potential width. The K(+) channel blocker 4-aminopyridine (10-50 μM) blocked rapidly activating, moderately inactivating currents that were more prevalent in PZ calyces. α-Dendrotoxin, a selective blocker of KV1 channels, reduced outward currents in CZ calyces but not in PZ calyces. Regional variations in K(+) conductances may contribute to different firing responses in calyx afferents.