Mechanoelectrical Transduction Channels Research Articles

Mutations of TMC1 cause deafness by disrupting mechanoelectrical transduction.

Mutations of transmembrane channel-like 1 gene (TMC1) can cause dominant (DFNA36) or recessive (DFNB7/B11) deafness. In this article, we describe the characteristics of DFNA36 and DFNB7/B11 deafness, the features of the Tmc1 mutant mouse strains, and recent advances in our understanding of TMC1 function. Publications related to TMC1, DFNA36, or DFNB7/B11 were identified through PubMed. All affected DFNA36 subjects showed post-lingual, progressive, sensorineural hearing loss (HL), initially affecting high frequencies. In contrast, almost all affected DFNB7/B11 subjects demonstrated congenital or prelingual severe to profound sensorineural HL. The mouse Tmc1 gene also has dominant and recessive mutant alleles that cause HL in mutant strains, including Beethoven, deafness, and Tmc1 knockout mice. These mutant mice have been instrumental for revealing that Tmc1 and its closely related paralog Tmc2 are expressed in cochlear and vestibular hair cells, and are required for hair cell mechanoelectrical transduction (MET). Recent studies suggest that TMC1 and TMC2 may be components of the long-sought hair cell MET channel. TMC1 mutations disrupt hair cell MET.

Optical stimulation of zebrafish hair cells expressing channelrhodopsin-2.

Vertebrate hair cells are responsible for the high fidelity encoding of mechanical stimuli into trains of action potentials (spikes) in afferent neurons. Here, we generated a transgenic zebrafish line expressing Channelrhodopsin-2 (ChR2) under the control of the hair-cell specific myo6b promoter, in order to examine the role of the mechanoelectrical transduction (MET) channel in sensory encoding in afferent neurons. We performed in vivo recordings from afferent neurons of the zebrafish lateral line while activating hair cells with either mechanical stimuli from a waterjet or optical stimuli from flashes of ∼470-nm light. Comparison of the patterns of encoded spikes during 100-ms stimuli revealed no difference in mean first spike latency between the two modes of activation. However, there was a significant increase in the variability of first spike latency during optical stimulation as well as an increase in the mean number of spikes per stimulus. Next, we compared encoding of spikes during hair-cell stimulation at 10, 20, and 40-Hz. Consistent with the increased variability of first spike latency, we saw a significant decrease in the vector strength of phase-locked spiking during optical stimulation. These in vivo results support a physiological role for the MET channel in the high fidelity of first spike latency seen during encoding of mechanical sensory stimuli. Finally, we examined whether remote activation of hair cells via ChR2 activation was sufficient to elicit escape responses in free-swimming larvae. In transgenic larvae, 100-ms flashes of ∼470-nm light resulted in escape responses that occurred concomitantly with field recordings indicating Mauthner cell activity. Altogether, the myo6b:ChR2 transgenic line provides a platform to investigate hair-cell function and sensory encoding, hair-cell sensory input to the Mauthner cell, and the ability to remotely evoke behavior in free-swimming zebrafish.

Transduction without tip links in cochlear hair cells is mediated by ion channels with permeation properties distinct from those of the mechano-electrical transducer channel.

Tip links between adjacent stereocilia are believed to gate mechano-electrical transducer (MET) channels and mediate the electrical responses of sensory hair cells. We found that mouse auditory hair cells that lack tip links due to genetic mutations or exposure to the Ca(2+) chelator BAPTA can, however, still respond to mechanical stimuli. These MET currents have unusual properties and are predominantly of the opposite polarity relative to those measured when tip links are present. There are other striking differences, for example, the channels are usually all closed when the hair cell is not stimulated and the currents in response to strong stimuli can be substantially larger than normal. These anomalous MET currents can also be elicited early in development, before the onset of mechano-electrical transduction with normal response polarity. Current-voltage curves of the anomalous MET currents are linear and do not show the rectification characteristic of normal MET currents. The permeant MET channel blocker dihydrostreptomycin is two orders of magnitude less effective in blocking the anomalous MET currents. The findings suggest the presence of a large population of MET channels with pore properties that are distinct from those of normal MET channels. These channels are not gated by hair-bundle links and can be activated under a variety of conditions in which normal tip-link-mediated transduction is not operational.

Touch as a Matter of Fat: The Phospholipids and DEG/ENaC Channels Needed for Metazoan Touch Sensation

Mechano-electrical transduction (MeT) channels are activated by mechanical energy and conduct ions across cell membranes. Such channels enable somatosensory neurons in our skin, joints, and muscles to monitor external touch and self-movement and to detect noxious inputs that initiate pain sensation. They also enable visceral sensory neurons such as baroreceptors to regulate heart function on a beat-by-beat basis. Genetic dissection in roundworms, fruitflies, and mice has led to the identification of many of the proteins that form MeT channels, including members of the TRP family of cation channels, DEG/ENaC sodium channels, and Piezo cation channels. This talk will present evidence establishing that DEG/ENaC sodium channels play crucial roles in both touch and nociception (O'Hagan et al, Nat Neurosci, 11:543, 2005; Geffeney et al, Neuron 71:845, 2011), highlighting aspects of the biophysics of MeT channel activation shared across neurons, species, and protein partners. To learn more about the physical basis of MeT channel activation in living sensory neurons, we are investigating the role of fatty acids and lipids in this process. Through a combination of genetic dissection and chemical complementation, we have identified specific poly-unsaturated phospholipids essential for normal touch sensation. We have also investigated how such lipids contribute to membrane mechanics. Collectively, our results support a model in which mechanosensitivity is a function not only of the proteins that form eukaryotic MeT channels, but also of the surrounding plasma membrane. Support: NIH NS047715; EB006745; AHA and HFSP Postdoctoral Fellowships.

Phospholipids that Contain Polyunsaturated Fatty Acids Enhance Neuronal Cell Mechanics and Touch Sensation

Mechanoelectrical transduction (MeT) channels embedded in neuronal cell membranes are essential for touch and proprioception. Little is understood about the interplay between native MeT channels and membrane phospholipids, in part because few techniques are available for altering plasma membrane composition invivo. Here, we leverage genetic dissection, chemical complementation, and optogenetics to establish that arachidonic acid (AA), an omega-6 polyunsaturated fatty acid, enhances touch sensation and mechanoelectrical transduction activity while incorporated into membrane phospholipids in C.elegans touch receptor neurons (TRNs). Because dynamic force spectroscopy reveals that AA modulates the mechanical properties of TRN plasma membranes, we propose that this polyunsaturated fatty acid (PUFA) is needed for MeT channel activity. These findings establish that polyunsaturated phospholipids are crucial determinants of both the biochemistry and mechanics of mechanoreceptor neurons and reinforce the idea that sensory mechanotransduction in animals relies on a cellular machine composed of both proteins and membrane lipids.

Omega 6 Polyunsaturated Fatty Acid-Containing Phospholipids Enhance Neuronal Cell Mechanics and Touch in C. Elegans

Mechano-electrical transduction (MeT) channels embedded in neuronal cell membranes are essential for touch and proprioception. Little is understood about the interplay between native MeT channels and membrane phospholipids, in part because few techniques are available for altering plasma membrane composition in vivo. Here, we leverage genetic dissection, chemical complementation, and optogenetics to establish that arachidonic acid (AA), an omega 6 polyunsaturated fatty acid (PUFA), enhances touch sensation and mechanoelectrical transduction activity while incorporated into membrane phospholipids in C. elegans touch receptor neurons (TRNs). We found that arachidonic acid acts cell autonomously, since we show that enzymes needed for its synthesis are expressed in TRNs. We also established that the membrane viscoelastic properties of TRNs lacking omega 6 PUFAs are altered (i.e., membrane bending and viscosity), yielding less flexible membranes than wild type, as determined by atomic force microscopy (AFM) based single-tether extrusion. Our data suggest that the defect in touch sensation likely reflects a loss of mechanotransduction rather than lack of excitability or downstream signaling. These findings establish that polyunsaturated phospholipids are crucial determinants of both the biochemistry and mechanics of mechanoreceptor neurons and reinforce the idea that sensory mechanotransduction in animals relies on a cellular machine composed of both proteins and membrane lipids.

Functional biology of ion channels: a review

Over the past few decades, a great deal of attention has been focused on discovering the protein partners that form mechano- electrical transduction (MeT) channels in somatic mechanoreceptors. Two classes of ion channel proteins are leading candidates: amiloride-sensitive channel (ASCs) and transient receptor potential (TRP) channel proteins. Here, we surveyed the literature to establish that most, if not all mechanoreceptor neurons in mice express multiple ASC and TRP channel proteins. But, the landscape of ion channel co-expression in mechanoreceptor neurons is only beginning to be mapped. Future work aimed at refining such maps for mammalian mechanoreceptor neurons will be critical for deeper understanding. Also, each of these potential MeT channel subunits operates within a large company of other ion channel actors that increase the complexity, flexibility, and robustness of somatosensory neuron function. Recently, two additional classes of membrane proteins (Piezo and TMC) have been linked to mechano-transduction. This situation is likely to exist in other mechanoreceptor neurons, including those responsible for touch and pain sensation in mammals.

Adaptation of Mammalian Auditory Hair Cell Mechanotransduction Is Independent of Calcium Entry

Adaptation is a hallmark of hair cell mechanotransduction, extending the sensory hair bundle dynamic range while providing mechanical filtering of incoming sound. In hair cells responsive to low frequencies, two distinct adaptation mechanisms exist, a fast component of debatable origin and a slow myosin-based component. It is generally believed that Ca(2+) entry through mechano-electric transducer channels is required for both forms of adaptation. This study investigates the calcium dependence of adaptation in the mammalian auditory system. Recordings from rat cochlear hair cells demonstrate that altering Ca(2+) entry or internal Ca(2+) buffering has little effect on either adaptation kinetics or steady-state adaptation responses. Two additional findings include a voltage-dependent process and an extracellular Ca(2+) binding site, both modulating the resting open probability independent of adaptation. These data suggest that slow motor adaptation is negligible in mammalian auditory cells and that the remaining adaptation process is independent of calcium entry.

Transient Receptor Potential Melastatin 1: A Hair Cell Transduction Channel Candidate

Sound and head movements are perceived through sensory hair cells in the inner ear. Mounting evidence indicates that this process is initiated by the opening of mechanically sensitive calcium-permeable channels, also referred to as the mechanoelectrical transducer (MET) channels, reported to be around the tips of all but the tallest stereocilia. However, the identity of MET channel remains elusive. Literature suggests that the MET channel is a non-selective cation channel with a high Ca2+ permeability and ∼100 picosiemens conductance. These characteristics make members of the transient receptor potential (TRP) superfamily likely candidates for this role. One of these candidates is the transient receptor potential melastatin 1 protein (TRPM1), which is expressed in various cells types within the cochlea of the mouse including the hair cells. Recent studies demonstrate that mutations in the TRPM1 gene underlie the inherited retinal disease complete congenital stationary night blindness in humans and depolarizing bipolar cell dysfunction in the mouse retina, but auditory function was not assessed. Here we investigate the role of Trpm1 in hearing and as a possible hair cell MET channel using mice homozygous for the null allele of Trpm1 (Trpm1−/−) or a missense mutation in the pore domain of TRPM1 (Trpm1tvrm27/tvrm27). Hearing thresholds were evaluated in adult (4–5 months old) mice with auditory-evoked brain stem responses. Our data shows no statistically significant difference in hearing thresholds in Trpm1−/− or Trpm1tvrm27/tvrm27 mutants compared to littermate controls. Further, none of the mutant mice showed any sign of balance disorder, such as head bobbing or circling. These data suggest that TRPM1 is not essential for development of hearing or balance and it is unlikely that TRPM1 is a component of the hair cell MET channel.

Auditory Distortions: Origins and Functions

To enhance weak sounds while compressing the dynamic intensity range, auditory sensory cells amplify sound-induced vibrations in a nonlinear, intensity-dependent manner. In the course of this process, instantaneous waveform distortion is produced, with two conspicuous kinds of interwoven consequences, the introduction of new sound frequencies absent from the original stimuli, which are audible and detectable in the ear canal as otoacoustic emissions, and the possibility for an interfering sound to suppress the response to a probe tone, thereby enhancing contrast among frequency components. We review how the diverse manifestations of auditory nonlinearity originate in the gating principle of their mechanoelectrical transduction channels; how they depend on the coordinated opening of these ion channels ensured by connecting elements; and their links to the dynamic behavior of auditory sensory cells. This paper also reviews how the complex properties of waves traveling through the cochlea shape the manifestations of auditory nonlinearity. Examination methods based on the detection of distortions open noninvasive windows on the modes of activity of mechanosensitive structures in auditory sensory cells and on the distribution of sites of nonlinearity along the cochlear tonotopic axis, helpful for deciphering cochlear molecular physiology in hearing-impaired animal models. Otoacoustic emissions enable fast tests of peripheral sound processing in patients. The study of auditory distortions also contributes to the understanding of the perception of complex sounds.

A molecular level prototype for mechanoelectrical transducer in mammalian hair cells

The mechanoelectrical transducer (MET) is a crucial component of mammalian auditory system. The gating mechanism of the MET channel remains a puzzling issue, though there are many speculations, due to the lack of essential molecular building blocks. To understand the working principle of mammalian MET, we propose a molecular level prototype which constitutes a charged blocker, a realistic ion channel and its surrounding membrane. To validate the proposed prototype, we make use of a well-established ion channel theory, the Poisson-Nernst-Planck equations, for three-dimensional (3D) numerical simulations. A wide variety of model parameters, including bulk ion concentration, applied external voltage, blocker charge and blocker displacement, are explored to understand the basic function of the proposed MET prototype. We show that our prototype prediction of channel open probability in response to blocker relative displacement is in remarkable accordance with experimental observation of rat cochlea outer hair cells. Our results appear to suggest that tip links which connect hair bundles gate MET channels.

Arachidonic Acid is Essential for Normal Touch Sensation

Touch, proprioception, and blood pressure regulation rely on mechanoreceptor neurons and mechano-electrical transduction (MeT) channels to convert mechanical cues into electrical signals. The protein partners that form native MeT channels are known only for a small group of mechanoreceptor neurons, including the touch receptor neurons responsible for gentle body touch in C. elegans. Such MeT channels are formed from at least four membrane proteins: two amiloride-sensitive sodium channel proteins (MEC-4 and MEC-10) and two putative lipid binding protein subunits (MEC-2 and MEC-6). It remains unknown how touch activates MeT channels, but interactions between the channel and the lipid membrane are likely to be essential. To learn more about interactions between MeT channels and specific lipids, we utilized C. elegans strains deficient in the synthesis of long-chain, poly-unsaturated fatty acids (PUFAs) and identified arachidonic acid (AA) as the sole PUFA required for full touch sensitivity. Touch receptor neurons retain normal morphology in PUFA-deficient mutants and normal touch sensitivity can be restored by exogenous PUFAs. We will present evidence garnered from behavioral and optogenetic studies suggesting that long-chain PUFAs are needed to activate native MeT channels, but dispensable for subsequent events linking sensory signaling in touch receptor neurons to behavior. Our data suggest that AA and its non-metabolizable analog ETYA directly modulate the activity of the MeT channel.

Compound Heterozygosity of the Functionally Null <i>Cdh23<sup>v-ngt</sup></i> and Hypomorphic <i>Cdh23<sup>ahl</sup></i> Alleles Leads to Early-onset Progressive Hearing Loss in Mice

The waltzer (v) mouse mutant harbors a mutation in Cadherin 23 (Cdh23) and is a model for Usher syndrome type 1D, which is characterized by congenital deafness, vestibular dysfunction, and prepubertal onset of progressive retinitis pigmentosa. In mice, functionally null Cdh23 mutations affect stereociliary morphogenesis and the polarity of both cochlear and vestibular hair cells. In contrast, the murine Cdh23ahl allele, which harbors a hypomorphic mutation, causes an increase in susceptibility to age-related hearing loss in many inbred strains. We produced congenic mice by crossing mice carrying the v niigata (Cdh23v-ngt) null allele with mice carrying the hypomorphic Cdh23ahl allele on the C57BL/6J background, and we then analyzed the animals’ balance and hearing phenotypes. Although the Cdh23v-ngt/ahl compound heterozygous mice exhibited normal vestibular function, their hearing ability was abnormal: the mice exhibited higher thresholds of auditory brainstem response (ABR) and rapid age-dependent elevation of ABR thresholds compared with Cdh23ahl/ahl homozygous mice. We found that the stereocilia developed normally but were progressively disrupted in Cdh23v-ngt/ahl mice. In hair cells, CDH23 localizes to the tip links of stereocilia, which are thought to gate the mechanoelectrical transduction channels in hair cells. We hypothesize that the reduction of Cdh23 gene dosage in Cdh23v-ngt/ahl mice leads to the degeneration of stereocilia, which consequently reduces tip link tension. These findings indicate that CDH23 plays an important role in the maintenance of tip links during the aging process.

Voltage oscillations and response dynamics in a model of sensory hair cells

Sensory hair cells in auditory and vestibular organs rely on active mechanisms to achieve high sensitivity and frequency selectivity with respect to weak stimuli. Self-sustained oscillations in hair cells occur on two very different levels. First, the mechano-sensory hair bundle itself can undergo spontaneous mechanical oscillations. Second, self-sustained electric voltage oscillations across the membrane of the hair cell have been documented in the inner ear of lower vertebrates. The functional significance of these self-sustained voltage oscillations is currently unknown. We used a Hodgkin-Huxley type model of the baso-lateral ionic currents of bullfrog sacculus to study genesis of spontaneous voltage oscillation patterns and how the spontaneous oscillations shape the response of the hair cell to external mechanical stimuli. To examine the influence of inevitable fluctuations on the dynamical regimes, we included a stochastic transduction current originating in the Brownian motion of the hair bundle and channel noise arising due to the finite number of mechano-electrical transduction channels [1]. We determined the bifurcation structure of the model in terms of two important ionic conductances, associated with the inwardly rectifier (K1) and Ca2+-activated (BK) potassium currents. We found that for large values of BK conductance the system is either at equilibrium or exhibit tonic oscillations. For small values of BK and large values of K1 conductances the dynamics of the model shows diverse patterns of activity including quasi-periodic oscillations, large-amplitude periodic spikes, and bursts of spikes. In particular we found a peculiar transition to bursting through quasiperiodic oscillations with two independent frequencies corresponding to a 2D torus in the phase space of the system. Within small patches of parameter space at the transition from spiking to bursting and at the spike adding transition, voltage dynamics are chaotic. Furthermore, we showed that thermal fluctuations of mechano-electrical transduction current can lead to chaos in a wide area of parameter space. We found a high sensitivity and frequency selectivity for the regime of regular spontaneous oscillations in response to sinusoidal stimuli with frequencies f > 5 Hz. Hence, an oscillatory voltage compartment might constitute a biophysical implementation of a high-gain amplifier. Cells poised in the chaotic regime of low BK and high K1 conductances showed poor tuning, but provided a high sensitivity to low-frequency variations of external stimuli. In summary, this study shows that the electrical oscillator found in saccular hair cells contributes significantly to nonlinear amplification of external mechanical stimuli. This further supports the idea of nonlinear oscillators playing a crucial role in the operation of the inner ear.

Estimation of optimal insertion angle in a mammalian outer hair cell stereocilium

Optimal insertion angle of mammalian stereocilia is estimated from the finite element analysis of the tip motion of outer hair cells (OHCs) stereocilia. The OHC stereocilia motion in the acousticolateral system appears to result in the mechanoelectrical transduction channels. Deflection of the hair bundle towards the tallest row of stereocilia causes increased probability of opening of ion channels. In this work, we focus on one of the physical features of the OHC stereocilium, the initial insertion angle of the tallest row into the tectorial membrane (TM), and its effects on the stereocilia's deflection motion. A three-dimensional model was built for the tallest stereocilium and the TM at the region where the best frequency was 500Hz. The mechanical interactions between the embedded stereocilia and the TM have been implemented into the finite element simulation. We found that, the optimum insertion angle of the tallest stereocilium into the TM was 69.8°, where the stereocilium is maximally deflected. This quantity is consistent with the histological observation obtained from the literature.

Age-DependentIn VivoConversion of Mouse Cochlear Pillar and Deiters' Cells to Immature Hair Cells by Atoh1 Ectopic Expression

Unlike nonmammalian vertebrates, mammals cannot convert inner ear cochlear supporting cells (SCs) into sensory hair cells (HCs) after damage, thus causing permanent deafness. Here, we achieved in vivo conversion of two SC subtypes, pillar cells (PCs) and Deiters' cells (DCs), into HCs by inducing targeted expression of Atoh1 at neonatal and juvenile ages using novel mouse models. The conversion only occurred in ∼10% of PCs and DCs with ectopic Atoh1 expression and started with reactivation of endogenous Atoh1 followed by expression of 11 HC and synaptic markers, a process that took approximately 3 weeks in vivo. These new HCs resided in the outer HC region, formed stereocilia, contained mechanoelectrical transduction channels, and survived for >2 months in vivo; however, they surprisingly lacked prestin and oncomodulin expression and mature HC morphology. In contrast, adult PCs and DCs no longer responded to ectopic Atoh1 expression, even after outer HC damage. Finally, permanent Atoh1 expression in endogenous HCs did not affect prestin expression but caused cell loss of mature HCs. Together, our results demonstrate that in vivo conversion of PCs and DCs into immature HCs by Atoh1 is age dependent and resembles normal HC development. Therefore, combined expression of Atoh1 with additional factors holds therapeutic promise to convert PCs and DCs into functional HCs in vivo for regenerative purposes.

Permeation properties of the hair cell mechanotransducer channel provide insight into its molecular structure

Mechanoelectric transducer (MET) channels, located near stereocilia tips, are opened by deflecting the hair bundle of sensory hair cells. Defects in this process result in deafness. Despite this critical function, the molecular identity of MET channels remains a mystery. Inherent channel properties, particularly those associated with permeation, provide the backbone for the molecular identification of ion channels. Here, a novel channel rectification mechanism is identified, resulting in a reduced pore size at positive potentials. The apparent difference in pore dimensions results from Ca(2+) binding within the pore, occluding permeation. Driving force for permeation at hyperpolarized potentials is increased because Ca(2+) can more easily be removed from binding within the pore due to the presence of an electronegative external vestibule that dehydrates and concentrates permeating ions. Alterations in Ca(2+) binding may underlie tonotopic and Ca(2+)-dependent variations in channel conductance. This Ca(2+)-dependent rectification provides targets for identifying the molecular components of the MET channel.

Systemic aminoglycosides are trafficked via endolymph into cochlear hair cells.

Aminoglycoside antibiotics rapidly enter and kill cochlear hair cells via apical mechanoelectrical transduction (MET) channels in vitro. In vivo, it remains unknown whether systemically-administered aminoglycosides cross the blood-labyrinth barrier into endolymph and enter hair cells. Here we show, for the first time, that systemic aminoglycosides are trafficked across the blood-endolymph barrier and preferentially enter hair cells across their apical membranes. This trafficking route is predominant compared to uptake via hair cell basolateral membranes during perilymph infusion.

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.

Activated by Force

An amiloride-sensitive sodium channel transduces intense mechanical stimuli in Caenorhabditis elegans .