The effects of Tmc1 Beethoven mutation on mechanotransducer channel function in cochlear hair cells

Sound stimuli are converted into electrical signals via gating of mechano-electrical transducer (MT) channels in the hair cell stereociliary bundle. The molecular composition of the MT channel is still not fully established, although transmembrane channel–like protein isoform 1 (TMC1) may be one component. We found that in outer hair cells of Beethoven mice containing a M412K point mutation in TMC1, MT channels had a similar unitary conductance to that of wild-type channels but a reduced selectivity for Ca²⁺. The Ca²⁺-dependent adaptation that adjusts the operating range of the channel was also impaired in Beethoven mutants, with reduced shifts in the relationship between MT current and hair bundle displacement for adapting steps or after lowering extracellular Ca²⁺; these effects may be attributed to the channel’s reduced Ca²⁺ permeability. Moreover, the density of stereociliary CaATPase pumps for Ca²⁺ extrusion was decreased in the mutant. The results suggest that a major component of channel adaptation is regulated by changes in intracellular Ca²⁺. Consistent with this idea, the adaptive shift in the current–displacement relationship when hair bundles were bathed in endolymph-like Ca²⁺ saline was usually abolished by raising the intracellular Ca²⁺ concentration.     

Theoretical conditions for high-frequency hair bundle oscillations in auditory hair cells

Substantial evidence exists for spontaneous oscillations of hair cell stereociliary bundles in the lower vertebrate inner ear. Since the oscillations are larger than expected from Brownian motion, they must result from an active process in the stereociliary bundle suggested to underlie amplification of the sensory input as well as spontaneous otoacoustic emissions. However, their low frequency (<100 Hz) makes them unsuitable for amplification in birds and mammals that hear up to 5 kHz or higher. To examine the possibility of high-frequency oscillations, we used a finite-element model of the outer hair cell bundle incorporating previously measured mechanical parameters. Bundle motion was assumed to activate mechanotransducer channels according to the gating spring hypothesis, and the channels were regulated adaptively by Ca²⁺ binding. The model generated oscillations of freestanding bundles at 4 kHz whose sharpness of tuning depended on the mechanotransducer channel number and location, and the Ca²⁺ concentration. Entrainment of the oscillations by external stimuli was used to demonstrate nonlinear amplification. The oscillation frequency depended on channel parameters and was increased to 23 kHz principally by accelerating Ca²⁺ binding kinetics. Spontaneous oscillations persisted, becoming very narrow-band, when the hair bundle was loaded with a tectorial membrane mass.     

Developmental changes in the cochlear hair cell mechanotransducer channel and their regulation by transmembrane channel–like proteins

Vibration of the stereociliary bundles activates calcium-permeable mechanotransducer (MT) channels to initiate sound detection in cochlear hair cells. Different regions of the cochlea respond preferentially to different acoustic frequencies, with variation in the unitary conductance of the MT channels contributing to this tonotopic organization. Although the molecular identity of the MT channel remains uncertain, two members of the transmembrane channel–like family, Tmc1 and Tmc2, are crucial to hair cell mechanotransduction. We measured MT channel current amplitude and Ca²⁺ permeability along the cochlea’s longitudinal (tonotopic) axis during postnatal development of wild-type mice and mice lacking Tmc1 (Tmc1−/−) or Tmc2 (Tmc2−/−). In wild-type mice older than postnatal day (P) 4, MT current amplitude increased ∼1.5-fold from cochlear apex to base in outer hair cells (OHCs) but showed little change in inner hair cells (IHCs), a pattern apparent in mutant mice during the first postnatal week. After P7, the OHC MT current in Tmc1−/− (dn) mice declined to zero, consistent with their deafness phenotype. In wild-type mice before P6, the relative Ca²⁺ permeability, P_Ca, of the OHC MT channel decreased from cochlear apex to base. This gradient in P_Ca was not apparent in IHCs and disappeared after P7 in OHCs. In Tmc1−/− mice, P_Ca in basal OHCs was larger than that in wild-type mice (to equal that of apical OHCs), whereas in Tmc2−/−, P_Ca in apical and basal OHCs and IHCs was decreased compared with that in wild-type mice. We postulate that differences in Ca²⁺ permeability reflect different subunit compositions of the MT channel determined by expression of Tmc1 and Tmc2, with the latter conferring higher P_Ca in IHCs and immature apical OHCs. Changes in P_Ca with maturation are consistent with a developmental decrease in abundance of Tmc2 in OHCs but not in IHCs.     

Nonlinear mechanical responses of mouse cochlear hair bundles.

The stiffness of sensory hair bundles of both inner (IHC) and outer (OHC) hair cells was measured with calibrated silica fibres in mouse cochlear cultures to test the hypothesis that the mechanical properties of the hair bundle reflect processes underlying mechanotransduction. For OHCs, the displacement of the hair bundle relaxed with time constants of 6 ms for displacements which open transducer channels and 4 ms for displacements which close the channels. The corresponding values of the time constants for IHCs were 10 ms and 8 ms, respectively. A displacement-dependent change in the stiffness of the hair bundle was not observed when the bundle was displaced orthogonally to the direction of excitation. The stiffness of the hair bundle as a function of nanometre displacements from the resting position was remarkably nonlinear. The stiffness declined to a minimum from the resting stiffness by about 12% for OHCs and 20% for IHCs when the hair bundle was displaced by about 20 nm in the excitatory direction, and it increased by a similar amount when the bundle was displaced by 20 nm in the inhibitory direction. The displacement at which the stiffness reached a minimum was within the most sensitive region of the hair-cell transducer function (receptor potential as a function of hair-bundle displacement), and the displacement at which the stiffness reached a maximum was at the point of saturation of the transducer function in the inhibitory direction. The nonlinear displacement-dependent compliance change is reversibly abolished, and the time constant of relaxation of the bundle for excitatory displacements is reversibly reduced, when mechanotransduction is blocked by the addition of either neomycin sulphate or cobalt chloride to the solution bathing the hair cells. The displacement-dependent compliance change was not apparently reduced when the receptor potential was attenuated through the substitution of sodium in the bathing solution with a less permeant cation, tetraethylammonium. These findings suggest that the nonlinear mechanical properties of the hair bundle are associated with aspects of the hair-cell mechanotransducer process. The mechanical properties of the hair bundle are discussed in relation to the 'gating-spring' hypothesis of hair-cell transduction.     

Conductance and block of hair-cell mechanotransducer channels in transmembrane channel–like protein mutants

Transmembrane channel–like (TMC) proteins TMC1 and TMC2 are crucial to the function of the mechanotransducer (MT) channel of inner ear hair cells, but their precise function has been controversial. To provide more insight, we characterized single MT channels in cochlear hair cells from wild-type mice and mice with mutations in Tmc1, Tmc2, or both. Channels were recorded in whole-cell mode after tip link destruction with BAPTA or after attenuating the MT current with GsMTx-4, a peptide toxin we found to block the channels with high affinity. In both cases, the MT channels in outer hair cells (OHCs) of wild-type mice displayed a tonotopic gradient in conductance, with channels from the cochlear base having a conductance (110 pS) nearly twice that of those at the apex (62 pS). This gradient was absent, with channels at both cochlear locations having similar small conductances, with two different Tmc1 mutations. The conductance of MT channels in inner hair cells was invariant with cochlear location but, as in OHCs, was reduced in either Tmc1 mutant. The gradient of OHC conductance also disappeared in Tmc1/Tmc2 double mutants, in which a mechanically sensitive current could be activated by anomalous negative displacements of the hair bundle. This “reversed stimulus–polarity” current was seen with two different Tmc1/Tmc2 double mutants, and with Tmc1/Tmc2/Tmc3 triple mutants, and had a pharmacological sensitivity comparable to that of native MT currents for most antagonists, except dihydrostreptomycin, for which the affinity was less, and for curare, which exhibited incomplete block. The existence in the Tmc1/Tmc2 double mutants of MT channels with most properties resembling those of wild-type channels indicates that proteins other than TMCs must be part of the channel pore. We suggest that an external vestibule of the MT channel may partly account for the channel’s large unitary conductance, high Ca²⁺ permeability, and pharmacological profile, and that this vestibule is disrupted in Tmc mutants.     

Amiloride causes changes in the mechanical properties of hair cell bundles in the fish lateral line similar to those induced by dihydrostreptomycin

Amiloride is a known blocker of the mechano-electrical transduction current in sensory hair cells. Measurements of cupular motion in the lateral line organ of fish now show that amiloride concurrently changes the micromechanical properties of the hair cell bundles. The effects of amiloride on the mechanics and receptor potentials of the hair cells resemble those previously observed for the aminoglycoside drug dihydrostreptomycin (DHSM) and are similarly antagonized by Ca²⁺. We hypothesize that amiloride and DHSM act on hair cells in two correlated ways which manifest themselves in both the electrical and mechanical properties of the transduction process. One action is the reduction of the transduction current with a concurrent increase of the hair bundle stiffness. The other action is a shift of the hair cell''s operating point on a current–displacement curve, with a concomitant shift along the associated hair bundle stiffness–displacement curve. The latter action has the opposite effect to that of the first and thus may lead, at relatively low blocker concentrations, to both an increase of transduction current and a decrease in hair bundle stiffness.     

Cell biology and genetics of root hair formation inArabidopsis thaliana

Summary In this review we integrate the information available on the cell biology of root hair formation with recent findings from the analysis of root hair mutants ofArabidopsis thaliana. The mature Arabidopsis root epidermis consists of root-hair-producing cells and non-root-hair-producing cells. Root hair growth begins with a swelling of the outer epidermal wall. It has been postulated that this is due to a pH-mediated localised cell wall loosening. From the bulge a single root hair emerges which grows by tip growth. The root hair tip consists of a vesicle-rich zone and an organelle-rich subapical zone. The vesicles supply new plasma membrane and cell wall material for elongation. The cytoskeleton and its associated regulatory proteins such as profilin and spectrin are proposed to be involved in the targeting of vesicles. Ca²⁺ influxes and gradients are present in hair tips, but their function is still unclear. Mutants have been isolated with lesions in various parts of the root hair developmental pathway from bulge identity and initiation, to control of tip diameter and extent and polarity of elongation.Abbreviations [Ca²⁺]_c cytosolic calcium concentration - MT microtubule - PM plasma membrane - VRZ vesicle-rich zone - WT wild type Dedicated to Professor Brian E. S. Gunning on the occasion of his 65th birthday     

The interaction of CaM7 and CNGC14 regulates root hair growth in Arabidopsis

Oscillations in cytosolic free calcium determine the polarity of tip‐growing root hairs. The Ca²⁺ channel cyclic nucleotide gated channel 14 (CNGC14) contributes to the dynamic changes in Ca²⁺ concentration gradient at the root hair tip. However, the mechanisms that regulate CNGC14 are unknown. In this study, we detected a direct interaction between calmodulin 7 (CaM7) and CNGC14 through yeast two‐hybrid and bimolecular fluorescence complementation assays. We demonstrated that the third EF‐hand domain of CaM7 specifically interacts with the cytosolic C‐terminal domain of CNGC14. A two‐electrode voltage clamp assay showed that CaM7 completely inhibits CNGC14‐mediated Ca²⁺ influx, suggesting that CaM7 negatively regulates CNGC14‐mediated calcium signaling. Furthermore, CaM7 overexpressing lines phenocopy the short root hair phenotype of a cngc14 mutant and this phenotype is insensitive to changes in external Ca²⁺ concentrations. We, thus, identified CaM7‐CNGC14 as a novel interacting module that regulates polar growth in root hairs by controlling the tip‐focused Ca²⁺ signal.     

Developmental regulation of TRPC3 ion channel expression in the mouse cochlea

Canonical transient receptor potential type 3 (TRPC3) ion channels assemble from TRPC3 subunits and exhibit multiple activation mechanisms. TRPC3 has been proposed to contribute to Ca²⁺ entry supporting Ca²⁺ homeostasis in cochlear hair cells and to be activated by G protein-coupled receptor (GPCR) signaling in spiral ganglion neurons. The present study was designed to determine the spatiotemporal profile of TRPC3 expression during mouse cochlear ontogeny. TRPC3 immunofluorescence of cryosectioned cochleae was performed using E16–adult tissue. We found that prior to birth, TRPC3 expression was strongest in epithelial cells that form the cochlear partition. In the early postnatal period, to the onset of hearing (~P12), immunofluorescence was strongest in the hair cells, with increased expression in stria vascularis and Reissner’s membrane. Afferent neurite labeling in inner spiral plexus and outer spiral bundles developed transiently in the perinatal period, corresponding to the critical period of synaptic consolidation, while signal in the spiral ganglion soma increased from the perinatal period through to adulthood. Compared with the late embryonic/early postnatal levels, hair cell expression was relatively weaker from the third postnatal week, whereas spiral ganglion soma labeling was stronger. In the adult, TRPC3 expression was primarily in the soma of spiral ganglion neurons, the hair cells, and the inner and outer sulcus regions. This spatiotemporal profile of TRPC3 expression was consistent with this ion channel contributing to development of sensory, neural and epithelial cochlear tissues, as well as hair cell Ca²⁺ homeostasis and regulation of auditory neurotransmission via GPCR signaling.     

TRPC3 ion channel subunit immunolocalization in the cochlea

Canonical transient receptor potential (TRPC) subunits assemble as tetramers to form ion channels with high calcium (Ca²⁺) permeability. Here, we investigated the possibility that TRPC3 ion channels are broadly expressed in the adult guinea pig and mouse cochleae. Using immunofluorescence, pronounced labeling occurred in the spiral ganglion (SG) neurons, inner hair cells (IHC), outer hair cells (OHC) and epithelial cells lining scala media. TRPC3 expression was homogeneous in the SG throughout the cochlea. In contrast, there was marked spatial variation in the immunolabeling in the cochlear hair cells with respect to location. This likely relates to the tonotopy of these cells. TRPC3 immunolabeling was more pronounced in the IHC than OHC. Both basal region IHC and OHC had higher TRPC3 expression levels than the corresponding cells from the apical region of the cochlea. These data suggest that TRPC3 ion channels contribute to Ca²⁺ homeostasis associated with the hair cells, with higher ion fluxes in more basal regions of the cochlea, and may also be a significant pathway for Ca²⁺ entry associated with auditory neurotransmission via the SG neurons. TRPC3 expression was also identified within the spiral limbus region, inner and outer sulcus, but without evidence for spatial variation in expression level. Expression in these gap junction-coupled epithelial cells lining scala media is indicative of a contribution of TRPC3 channels to cochlear electrochemical homeostasis.     

ATPase activity of myosin in hair bundles of the bullfrog''s sacculus.

Mechanoelectrical transduction by a hair cell displays adaptation, which is thought to occur as myosin-based molecular motors within the mechanically sensitive hair bundle adjust the tension transmitted to transduction channels. To assess the enzymatic capabilities of the myosin isozymes in hair bundles, we examined the actin-dependent ATPase activity of bundles isolated from the bullfrog's sacculus. Separation of 32P-labeled inorganic phosphate from unreacted [gamma-32P]ATP by thin-layer chromatography enabled us to measure the liberation of as little as 0.1 fmol phosphate. To distinguish the Mg(2+)-ATPase activity of myosin isozymes from that of other hair-bundle enzymes, we inhibited the interaction of hair-bundle myosin with actin and determined the reduction in ATPase activity. N-ethylmaleimide (NEM) decreased neither physiologically measured adaptation nor the nucleotide-hydrolytic activity of a 120-kDa protein thought to be myosin 1 beta. The NEM-insensitive, actin-activated ATPase activity of myosin increased from 1.0 fmol x s-1 in 1 mM EGTA to 2.3 fmol x s-1 in 10 microM Ca2+. This activity was largely inhibited by calmidazolium, but was unaffected by the addition of exogenous calmodulin. These results, which indicate that hair bundles contain enzymatically active, Ca(2+)-sensitive myosin molecules, are consistent with the role of Ca2+ in adaptation and with the hypothesis that myosin forms the hair cell's adaptation motor.     

A mechanism for active hearing

The remarkable sensitivity, frequency selectivity, and nonlinearity of the cochlea have been attributed to the putative 'cochlear amplifier', which consumes metabolic energy to amplify the cochlear mechanical response to sounds. Recent studies have demonstrated that outer hair cells actively generate force using somatic electromotility and active hair-bundle motion. However, the expected power gain of the cochlear amplifier has not been demonstrated experimentally, and the measured location of cochlear nonlinearity is inconsistent with the predicted location of the cochlear amplifier. We instead propose a 'cochlear transformer' mechanism to interpret cochlear performance.     

A quantitative comparison of mechanoelectrical transduction in vestibular and auditory hair cells of neonatal mice.

Vestibular hair cells (VHCs) and cochlear outer hair cells (OHCs) of neonatal mice were stimulated by a fluid jet directed at their stereociliary bundles. Relations between the force exerted by the jet, bundle displacement, and the resulting transducer current were studied. The mean maximum transducer conductance in VHCs (2.6 nS) was about half that of the OHCs (5.5 nS), with the largest recorded values being 4.1 nS and 9.2 nS, respectively. In some OHCs activity of a single, 112 pS transducer channel was observed, allowing an estimate of the maximum number of channels: up to 36 in VHCs and 82 in OHCs, corresponding to about one transducer channel per tip link. The VHC bundles required about 330 nm of tip displacement to activate 90% of the maximum transducer conductance, compared to 150 nm for the OHC bundles. This corresponded to 2 deg of rotation about their pivots for both, due to the greater length of the VHC bundles. The VHC bundles'' translational stiffness was one-seventh of that of the OHCs. Conversion to rotational stiffness almost abolished this difference. Rotation of the hair bundle rather than translation determines the gating of the transducer channels, independent of bundle height or origin of the cells.     

Mitochondria modulate the spatio-temporal properties of intra- and intercellular Ca signals in cochlear supporting cells

In the cochlea, cell damage triggers intercellular Ca²⁺ waves that propagate through the glial-like supporting cells that surround receptor hair cells. These Ca²⁺ waves are thought to convey information about sensory hair cell-damage to the surrounding supporting cells within the cochlear epithelium. Mitochondria are key regulators of cytoplasmic Ca²⁺ concentration ([Ca²⁺]_cyt), and yet little is known about their role during the propagation of such intercellular Ca²⁺ signalling. Using neonatal rat cochlear explants and fluorescence imaging techniques, we explore how mitochondria modulate supporting cell [Ca²⁺]_cyt signals that are triggered by ATP or by hair cell damage. ATP application (0.1–50 μM) caused a dose dependent increase in [Ca²⁺]_cyt which was accompanied by an increase in mitochondrial calcium. Blocking mitochondrial Ca²⁺ uptake by dissipating the mitochondrial membrane potential using CCCP and oligomycin or using Ru360, an inhibitor of the mitochondrial Ca²⁺ uniporter, enhanced the peak amplitude and duration of ATP-induced [Ca²⁺]_cyt transients. In the presence of Ru360, the mean propagation velocity, amplitude and extent of spread of damage-induced intercellular Ca²⁺ waves was significantly increased. Thus, mitochondria function as spatial Ca²⁺ buffers during agonist-evoked [Ca²⁺]_cyt signalling in cochlear supporting cells and play a significant role in regulating the spatio-temporal properties of intercellular Ca²⁺ waves.     

Response to a pure tone in a nonlinear mechanical-electrical-acoustical model of the cochlea

In this article, a nonlinear mathematical model is developed based on the physiology of the cochlea of the guinea pig. The three-dimensional intracochlear fluid dynamics are coupled to a micromechanical model of the organ of Corti and to electrical potentials in the cochlear ducts and outer hair cells (OHC). OHC somatic electromotility is modeled by linearized piezoelectric relations whereas the OHC hair-bundle mechanoelectrical transduction current is modeled as a nonlinear function of the hair-bundle deflection. The steady-state response of the cochlea to a single tone is simulated in the frequency domain using an alternating frequency time scheme. Compressive nonlinearity, harmonic distortion, and DC shift on the basilar membrane (BM), tectorial membrane (TM), and OHC potentials are predicted using a single set of parameters. The predictions of the model are verified by comparing simulations to available in vivo experimental data for basal cochlear mechanics. In particular, the model predicts more amplification on the reticular lamina (RL) side of the cochlear partition than on the BM, which replicates recent measurements. Moreover, small harmonic distortion and DC shifts are predicted on the BM, whereas more significant harmonic distortion and DC shifts are predicted in the RL and TM displacements and in the OHC potentials.     

Making an effort to listen: mechanical amplification in the ear

The inner ear's performance is greatly enhanced by an active process defined by four features: amplification, frequency selectivity, compressive nonlinearity, and spontaneous otoacoustic emission. These characteristics emerge naturally if the mechanoelectrical transduction process operates near a dynamical instability, the Hopf bifurcation, whose mathematical properties account for specific aspects of our hearing. The active process of nonmammalian tetrapods depends upon active hair-bundle motility, which emerges from the interaction of negative hair-bundle stiffness and myosin-based adaptation motors. Taken together, these phenomena explain the four characteristics of the ear's active process. In the high-frequency region of the mammalian cochlea, the active process is dominated instead by the phenomenon of electromotility, in which the cell bodies of outer hair cells extend and contract as the protein prestin alters its membrane surface area in response to changes in membrane potential.     

Divalent counterions tether membrane-bound carbohydrates to promote the cohesion of auditory hair bundles

The cell membranes in the hair bundle of an auditory hair cell confront a difficult task as the bundle oscillates in response to sound: for efficient mechanotransduction, all the component stereocilia of the hair bundle must move essentially in unison, shearing at their tips yet maintaining contact without membrane fusion. One mechanism by which this cohesion might occur is counterion-mediated attachment between glycan components of apposed stereociliary membranes. Using capillary electrophoresis, we showed that the stereociliary glycocalyx acts as a negatively charged polymer brush. We found by force-sensing photomicrometry that the stereocilia formed elastic connections with one another to various degrees depending on the surrounding ionic environment and the presence of N-linked sugars. Mg²⁺ was a more potent mediator of attachment than was Ca²⁺. The forces between stereocilia produced chaotic stick-slip behavior. These results indicate that counterion-mediated interactions in the glycocalyx contribute to the stereociliary coherence that is essential for hearing.     

Effectiveness of Hair Bundle Motility as the Cochlear Amplifier

The effectiveness of hair bundle motility in mammalian and avian ears is studied by examining energy balance for a small sinusoidal displacement of the hair bundle. The condition that the energy generated by a hair bundle must be greater than energy loss due to the shear in the subtectorial gap per hair bundle leads to a limiting frequency that can be supported by hair-bundle motility. Limiting frequencies are obtained for two motile mechanisms for fast adaptation, the channel re-closure model and a model that assumes that fast adaptation is an interplay between gating of the channel and the myosin motor. The limiting frequency obtained for each of these models is an increasing function of a factor that is determined by the morphology of hair bundles and the cochlea. Primarily due to the higher density of hair cells in the avian inner ear, this factor is ∼10-fold greater for the avian ear than the mammalian ear, which has much higher auditory frequency limit. This result is consistent with a much greater significance of hair bundle motility in the avian ear than that in the mammalian ear.