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Transmembrane channel-like protein 1 (TMC1) is thought to form the ion-conducting pore of the mechanoelectrical transducer (MET) channel in auditory hair cells. Using single-channel analysis and ionic permeability measurements, we characterized six missense mutations in the purported pore region of mouse TMC1. All mutations reduced the Ca 2+ permeability of the MET channel, triggering hair cell apoptosis and deafness. In addition, Tmc1 p.E520Q and Tmc1 p.D528N reduced channel conductance, whereas Tmc1 p.W554L and Tmc1 p.D569N lowered channel expression without affecting the conductance. Tmc1 p.M412K and Tmc1 p.T416K reduced only the Ca 2+ permeability. The consequences of these mutations endorse TMC1 as the pore of the MET channel. The accessory subunits, LHFPL5 and TMIE, are thought to be involved in targeting TMC1 to the tips of the stereocilia. We found sufficient expression of TMC1 in outer hair cells of Lhfpl5 and Tmie knockout mice to determine the properties of the channels, which could still be gated by hair bundle displacement. Single-channel conductance was unaffected in Lhfpl5 −/− but was reduced in Tmie −/− , implying TMIE very likely contributes to the pore. Both the working range and half-saturation point of the residual MET current in Lhfpl5 −/− were substantially increased, suggesting that LHFPL5 is part of the mechanical coupling between the tip-link and the MET channel. Based on counts of numbers of stereocilia per bundle, we estimate that each PCDH15 and LHFPL5 monomer may contact two channels irrespective of location.

Transmembrane channel-like protein isoform 1 (TMC1) is a major component of the mechano-electrical transducer (MET) channel in cochlear hair cells and is subject to numerous mutations causing deafness. We report a new dominant human deafness mutation,TMC1p.T422K, and have characterized the homologous mouse mutant,Tmc1p.T416K, which caused deafness and outer hair cell (OHC) loss by the fourth postnatal week. MET channels showed decreased Ca2+permeability and resting open probability, but no change in single-channel conductance or expression. Three adjacent deafness mutations areTMC1p.L416R, p.G417R, and p.M418K, the last homologous to the mouseBeethoventhat exhibits similar channel effects. All substitute a positive for a neutral residue, which could produce charge screening in the channel pore or influence binding of an accessory subunit. Channel properties were compared in mice of both sexes between dominant (Tmc1p.T416K,Tmc1p.D569N) and recessive (Tmc1p.W554L,Tmc1p.D528N) mutations of residues near the putative pore of the channel.Tmc1p.W554L and p.D569N exhibit reduced maximum current with no effect on single-channel conductance, implying a smaller number of channels transported to the stereociliary tips; this may stem from impaired TMC1 binding to LHFPL5.Tmc1p.D528N, located in the pore's narrowest region, uniquely caused large reductions in MET channel conductance and block by dihydrostreptomycin (DHS). ForTmc1p.T416K andTmc1p.D528N, transduction loss occurred between P15 and P20. We propose two mechanisms linking channel mutations and deafness: decreased Ca2+permeability, common to all mutants, and decreased resting open probability in low Ca2+, confined to dominant mutations.SIGNIFICANCE STATEMENTTransmembrane channel-like protein isoform 1 (TMC1) is thought to be a major component of the mechanotransducer channel in auditory hair cells, but the protein organization and channel structure are still uncertain. We made four mouse lines harboringTmc1point mutations that alter channel properties, causing hair cell degeneration and deafness. These include a mouse homolog of a new human deafness mutation pT416K that decreased channel Ca2+permeability by introducing a positively-charged amino acid in the putative pore. All mutations are consistent with the channel structure predicted from modeling, but only one, p.D528N near the external face of the pore, substantially reduced channel conductance and Ca2+permeability and virtually abolished block by dihydrostreptomycin (DHS), strongly endorsing its siting within the pore.

Beurg et al. report that the conductance of cochlear hair cell mechanotransducer channels inferred from single-channel events is larger than using noise analysis, which underestimates size due to filtering of fast openings. The difference leads to a first estimate of the channel activation time constant., 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.

Ototoxicity, noise overstimulation, or aging, can all produce hearing loss with similar properties, in which outer hair cells (OHCs), principally those at the high-frequency base of the cochlea, are preferentially affected. We suggest that the differential vulnerability may partly arise from differences in Ca(2+) balance among cochlear locations. Homeostasis is determined by three factors: Ca(2+) influx mainly via mechanotransducer (MET) channels; buffering by calcium-binding proteins and organelles like mitochondria; and extrusion by the plasma membrane CaATPase pump. We review quantification of these parameters and use our experimentally-determined values to model changes in cytoplasmic and mitochondrial Ca(2+) during Ca(2+) influx through the MET channels. We suggest that, in OHCs, there are two distinct micro-compartments for Ca(2+) handling, one in the hair bundle and the other in the cell soma. One conclusion of the modeling is that there is a tonotopic gradient in the ability of OHCs to handle the Ca(2+) load, which correlates with their vulnerability to environmental challenges. High-frequency basal OHCs are the most susceptible because they have much larger MET currents and have smaller dimensions than low-frequency apical OHCs.

Functional mechanoelectrical transduction (MET) channels of cochlear hair cells require the presence of transmembrane channel-like protein isoforms TMC1 or TMC2. We show that TMCs are required for normal stereociliary bundle development and distinctively influence channel properties. TMC1-dependent channels have larger single-channel conductance and in outer hair cells (OHCs) support a tonotopic apex-to-base conductance gradient. Each MET channel complex exhibits multiple conductance states in ~50 pS increments, basal MET channels having more large-conductance levels. Using mice expressing fluorescently tagged TMCs, we show a three-fold increase in number of TMC1 molecules per stereocilium tip from cochlear apex to base, mirroring the channel conductance gradient in OHCs. Single-molecule photobleaching indicates the number of TMC1 molecules per MET complex changes from ~8 at the apex to ~20 at base. The results suggest there are varying numbers of channels per MET complex, each requiring multiple TMC1 molecules, and together operating in a coordinated or cooperative manner., Mechanoelectrical transduction channel (MET) current found in stereocilia of hair cells matures over the first postnatal week. Here the authors look at the contribution of transmembrane channel-like protein 1 and 2 (TMC1 and TMC2) to MET current during development of tonotopic gradients.

Throughout postnatal maturation of the mouse inner ear, cochlear hair cells display at least two types of mechanically gated ion channel: normal mechanotransducer (MT) channels at the tips of the stereocilia, activated by tension in interciliary tip links, and anomalous mechanosensitive (MS) channels on the top surface of the cells. The anomalous MS channels are responsible for the reverse-polarity current that appears in mutants in which normal transduction is lost. They are also seen in wild-type hair cells around birth, appearing 2 days earlier than normal MT channels, and being down-regulated with the emergence of the normal channels. We review the evidence that the normal and anomalous channels are distinct channel types, which includes differences in localization, susceptibility to pharmacological agents, single-channel conductance and Ca2+ permeability. The dichotomy is reinforced by the observation that the anomalous current is absent in cochlear cells of Piezo2-null mice, even though the normal MT current persists. The anomalous current is suppressed by high intracellular Ca2+ , suggesting that influx of the divalent ion via more Ca2+ -permeable normal MT channels inhibits the anomalous channels, thus explaining the temporal relationship between the two. Piezo2-null mice have largely normal hearing, exhibiting up to 20 dB elevation in threshold in the acoustic brainstem response, so raising questions about the significance of PIEZO2 in the cochlea. Since the anomalous current declines with postnatal age, PIEZO2 may contribute to hair cell development, but it does not underlie the normal MT current. Its role in the development of hearing is not understood.

Sound pressure fluctuations striking the ear are conveyed to the cochlea, where they vibrate the basilar membrane on which sit hair cells, the mechanoreceptors of the inner ear. Recordings of hair cell electrical responses have shown that they transduce sound via submicrometer deflections of their hair bundles, which are arrays of interconnected stereocilia containing the mechanoelectrical transducer (MET) channels. MET channels are activated by tension in extracellular tip links bridging adjacent stereocilia, and they can respond within microseconds to nanometer displacements of the bundle, facilitated by multiple processes of Ca2+-dependent adaptation. Studies of mouse mutants have produced much detail about the molecular organization of the stereocilia, the tip links and their attachment sites, and the MET channels localized to the lower end of each tip link. The mammalian cochlea contains two categories of hair cells. Inner hair cells relay acoustic information via multiple ribbon synapses that transmit rapidly without rundown. Outer hair cells are important for amplifying sound-evoked vibrations. The amplification mechanism primarily involves contractions of the outer hair cells, which are driven by changes in membrane potential and mediated by prestin, a motor protein in the outer hair cell lateral membrane. Different sound frequencies are separated along the cochlea, with each hair cell being tuned to a narrow frequency range; amplification sharpens the frequency resolution and augments sensitivity 100-fold around the cell's characteristic frequency. Genetic mutations and environmental factors such as acoustic overstimulation cause hearing loss through irreversible damage to the hair cells or degeneration of inner hair cell synapses. © 2017 American Physiological Society. Compr Physiol 7:1197-1227, 2017.

Discrimination of different sound frequencies is pivotal to recognizing and localizing friend and foe. Here, I review the various hair-cell tuning mechanisms employed among vertebrates. Electrical resonance, filtering of the receptor potential by voltage-dependent ion channels, is ubiquitous in all non-mammals, but has an upper limit of about 1 kHz. The frequency range is extended by mechanical resonance of the hair bundles in frogs and lizards, but may need active hair-bundle motion to achieve sharp tuning up to 5 kHz. Tuning in mammals employs somatic motility of outer hair cells, underpinned by the membrane protein prestin, to expand the frequency range. The bird cochlea may also employ prestin at high frequencies, but hair cells below 1 kHz show electrical resonance.

Significance Cochlear hair cells transduce sound into electrical signals by activation of mechanically sensitive ion channels thought to be formed by TMC1. We generated a single aspartate/asparagine substitution in mouse TMC1 which is homologous to a human genetic deafness mutation. The main consequence was reduction in the Ca2+ permeability of the mechanically sensitive channel with little change in its unitary conductance. Nevertheless, there was a much reduced expression of the ion channel, which led within 4 wk to death of the outer hair cells culminating in deafness. The mouse mutant accounts for the human deafness and implies that TMC1, in addition to forming the mechanically sensitive ion channel, regulates its own expression., Mechanoelectrical transducer (MET) currents were recorded from cochlear hair cells in mice with mutations of transmembrane channel-like protein TMC1 to study the effects on MET channel properties. We characterized a Tmc1 mouse with a single-amino-acid mutation (D569N), homologous to a dominant human deafness mutation. Measurements were made in both Tmc2 wild-type and Tmc2 knockout mice. By 30 d, Tmc1 pD569N heterozygote mice were profoundly deaf, and there was substantial loss of outer hair cells (OHCs). MET current in OHCs of Tmc1 pD569N mutants developed over the first neonatal week to attain a maximum amplitude one-third the size of that in Tmc1 wild-type mice, similar at apex and base, and lacking the tonotopic size gradient seen in wild type. The MET-channel Ca2+ permeability was reduced 3-fold in Tmc1 pD569N homozygotes, intermediate deficits being seen in heterozygotes. Reduced Ca2+ permeability resembled that of the Tmc1 pM412K Beethoven mutant, a previously studied semidominant mouse mutation. The MET channel unitary conductance, assayed by single-channel recordings and by measurements of current noise, was unaffected in mutant apical OHCs. We show that, in contrast to the Tmc1 M412K mutant, there was reduced expression of the TMC1 D569N channel at the transduction site assessed by immunolabeling, despite the persistence of tip links. The reduction in MET channel Ca2+ permeability seen in both mutants may be the proximate cause of hair-cell apoptosis, but changes in bundle shape and protein expression in Tmc1 D569N suggest another role for TMC1 apart from forming the channel.

Key points Hair cell mechanoelectrical transducer channels are opened by deflections of the hair bundle about a resting position set by incompletely understood adaptation mechanisms.We used three characteristics to define adaptation in hair cell mutants of transmembrane channel‐like proteins, TMC1 and TMC2, which are considered to be channel constituents.The results obtained demonstrate that the three characteristics are not equivalent, and raise doubts about simple models in which intracellular Ca2+ regulates adaptation.Adaptation is faster and more effective in TMC1‐containing than in TMC2‐containing transducer channels. This result ties adaptation to the channel complex, and suggests that TMC1 is a better isoform for use in cochlear hair cells.We describe a TMC1 point mutation, D569N, that reduces the resting open probability and Ca2+ permeability of the transducer channels, comprising properties that may contribute to the deafness phenotype. Abstract Recordings of mechanoelectrical transducer (MET) currents in cochlear hair cells were made in mice with mutations of transmembrane channel‐like (TMC) protein to examine the effects on fast transducer adaptation. Adaptation was faster and more complete in Tmc2–/– than in Tmc1–/–, although this disparity was not explained by differences in Ca2+ permeability or Ca2+ influx between the two isoforms, with TMC2 having the larger permeability. We made a mouse mutation, Tmc1 p.D569N, homologous to a human DFNA36 deafness mutation, which also had MET channels with lower Ca2+‐permeability but showed better fast adaptation than wild‐type Tmc1+/+ channels. Consistent with the more effective adaptation in Tmc1 p.D569N, the resting probability of MET channel opening was smaller. The three TMC variants studied have comparable single‐channel conductances, although the lack of correlation between channel Ca2+ permeability and adaptation opposes the hypothesis that adaptation is controlled simply by Ca2+ influx through the channels. During the first postnatal week of mouse development, the MET currents amplitude grew, and transducer adaptation became faster and more effective. We attribute changes in adaptation partly to a developmental switch from TMC2‐ to TMC1‐ containing channels and partly to an increase in channel expression. More complete and faster adaptation, coupled with larger MET currents, may account for the sole use of TMC1 in the adult cochlear hair cells., Key points Hair cell mechanoelectrical transducer channels are opened by deflections of the hair bundle about a resting position set by incompletely understood adaptation mechanisms.We used three characteristics to define adaptation in hair cell mutants of transmembrane channel‐like proteins, TMC1 and TMC2, which are considered to be channel constituents.The results obtained demonstrate that the three characteristics are not equivalent, and raise doubts about simple models in which intracellular Ca2+ regulates adaptation.Adaptation is faster and more effective in TMC1‐containing than in TMC2‐containing transducer channels. This result ties adaptation to the channel complex, and suggests that TMC1 is a better isoform for use in cochlear hair cells.We describe a TMC1 point mutation, D569N, that reduces the resting open probability and Ca2+ permeability of the transducer channels, comprising properties that may contribute to the deafness phenotype.

Adult stem cells are undifferentiated cells with the capacity to proliferate and form mature tissue-specific cell types. Nestin is an intermediate filament protein used to identify cells with stem cell characteristics. Its expression has been observed in a population of cells in developing and adult cochleae. In vitro studies using rodent cochlear tissue have documented the potential of nestin-expressing cells to proliferate and form hair and supporting cells. In this study, nestin coupled to green fluorescent protein (GFP) transgenic mice were used to provide a more complete characterization of the spatial and temporal expression of nestin in the inner ear, from organogenesis to adulthood. During development, nestin is expressed in the spiral ganglion cell region and in multiple cell types in the organ of Corti, including nascent hair and supporting cells. In adulthood, its expression is reduced but persists in the spiral ganglion, in a cell population medial to and below the inner hair cells, and in Deiters' cells in the cochlear apex. Moreover, nestin-expressing cells can proliferate in restricted regions of the inner ear during development shown by coexpression with Ki67 and MCM2 and by 5-ethynyl-2′-deoxyuridine incorporation. Results suggest that nestin may label progenitor cells during inner ear development and may not be a stem cell marker in the mature organ of Corti; however, nestin-positive cells in the spiral ganglion exhibit some stem cell characteristics. Future studies are necessary to determine if these cells possess any latent stem cell-like qualities that may be targeted as a regenerative approach to treat neuronal forms of hearing loss.

Transmembrane channel-like protein isoform-1 (TMC1) has emerged over the past five years as a prime contender for the mechano-electrical transducer (MET) channel in hair cells of the inner ear. TMC1 is thought to have a six-transmembrane domain structure reminiscent of some other ion-channel subunits, and is targeted to the tips of the stereocilia in the sensory hair bundle, where the MET channel is located. Moreover, there are TMC1 mutations linked to human deafness causing loss of conventional MET currents, hair cell degeneration, and deafness in mice. Finally, mutations of Tmc1 can alter the conductance and Ca2+ selectivity of the MET channels. For several reasons though, it is unclear that TMC1 is indeed the MET channel pore: 1) in other animals or tissues, mutations of TMC family members do not directly affect cellular mechanosensitivity; 2) there are residual manifestations of mechanosensitivity in hair cells of mouse Tmc1:Tmc2 double knockouts; 3) there is so far no evidence that expression of mammalian Tmc1 generates a mechanically sensitive ion channel in the plasma membrane when expressed in heterologous cells; and 4) there are other proteins, such as TMIE and LHFPL5, which behave similarly to TMC1, their mutation also leading to loss of MET current and deafness. This review will present these disparate lines of evidence and describes recent work that addresses the role of TMC1.

Cochlear hair cells normally detect positive deflections of their hair bundles, rotating toward their tallest edge, which opens mechanotransducer (MT) channels by increased tension in interciliary tip links. After tip-link destruction, the normal polarity of MT current is replaced by a mechanically sensitive current evoked by negative bundle deflections. The "reverse-polarity" current was investigated in cochlear hair cells after tip-link destruction with BAPTA, in transmembrane channel-like protein isoforms 1/2 (Tmc1:Tmc2) double mutants, and during perinatal development. This current is a natural adjunct of embryonic development, present in all wild-type hair cells but declining after birth with emergence of the normal-polarity current. Evidence indicated the reverse-polarity current seen developmentally was a manifestation of the same ion channel as that evident under abnormal conditions in Tmc mutants or after tip-link destruction. In all cases, sinusoidal fluid-jet stimuli from different orientations suggested the underlying channels were opened not directly by deflections of the hair bundle but by deformation of the apical plasma membrane. Cell-attached patch recording on the hair-cell apical membrane revealed, after BAPTA treatment or during perinatal development, 90-pS stretch-activated cation channels that could be blocked by Ca(2+) and by FM1-43. High-speed Ca(2+) imaging, using swept-field confocal microscopy, showed the Ca(2+) influx through the reverse-polarity channels was not localized to the hair bundle, but distributed across the apical plasma membrane. These reverse-polarity channels, which we propose to be renamed "unconventional" mechanically sensitive channels, have some properties similar to the normal MT channels, but the relationship between the two types is still not well defined.

Analyses of the Tmc1 Beethoven mouse mutant indicate that hair cell mechanotransducer channel adaptation in mammals is mainly regulated by changes in intracellular Ca2+., 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.

Proteins other than TMC1 and TMC2 must contribute to the pore of the mechanotransducer channel of cochlear hair cells; an external vestibule subject to disruption in Tmc mutants may influence the channel’s properties., 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.

Inner ear hair cells detect sound through deflection of stereocilia, the microvilli-like projections that are arranged in rows of graded heights. Calcium and integrin-binding protein 2 is essential for hearing and localizes to stereocilia, but its exact function is unknown. Here, we have characterized two mutant mouse lines, one lacking calcium and integrin-binding protein 2 and one carrying a human deafness-related Cib2 mutation, and show that both are deaf and exhibit no mechanotransduction in auditory hair cells, despite the presence of tip links that gate the mechanotransducer channels. In addition, mechanotransducing shorter row stereocilia overgrow in hair cell bundles of both Cib2 mutants. Furthermore, we report that calcium and integrin-binding protein 2 binds to the components of the hair cell mechanotransduction complex, TMC1 and TMC2, and these interactions are disrupted by deafness-causing Cib2 mutations. We conclude that calcium and integrin-binding protein 2 is required for normal operation of the mechanotransducer channels and is involved in limiting the growth of transducing stereocilia., Inner ear hair cells detect sound through deflection of stereocilia that harbor mechanically-gated channels. Here the authors show that protein responsible for Usher syndrome, CIB2, interacts with these channels and is essential for their function and hearing in mice.

Sound stimuli elicit movement of the stereocilia that make up the hair bundle of cochlear hair cells, putting tension on the tip links connecting the stereocilia and thereby opening mechanotransducer (MT) channels. Tmc1 and Tmc2, two members of the transmembrane channel–like family, are necessary for mechanotransduction. To assess their precise role, we recorded MT currents elicited by hair bundle deflections in mice with null mutations of Tmc1, Tmc2, or both. During the first postnatal week, we observed a normal MT current in hair cells lacking Tmc1 or Tmc2; however, in the absence of both isoforms, we recorded a large MT current that was phase-shifted 180°, being evoked by displacements of the hair bundle away from its tallest edge rather than toward it as in wild-type hair cells. The anomalous MT current in hair cells lacking Tmc1 and Tmc2 was blocked by FM1-43, dihydrostreptomycin, and extracellular Ca2+ at concentrations similar to those that blocked wild type. MT channels in the double knockouts carried Ca2+ with a lower permeability than wild-type or single mutants. The MT current in double knockouts persisted during exposure to submicromolar Ca2+, even though this treatment destroyed the tip links. We conclude that the Tmc isoforms do not themselves constitute the MT channel but are essential for targeting and interaction with the tip link. Changes in the MT conductance and Ca2+ permeability observed in the absence of Tmc1 mutants may stem from loss of interaction with protein partners in the transduction complex.

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 Ca(2+) 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 Ca(2+) 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 Ca(2+) 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.

Auditory hair cells contain mechanotransduction channels that rapidly open in response to sound-induced vibrations. Surprisingly, we report here that auditory hair cells contain two molecularly distinct mechanotransduction channels. One ion channel is activated by sound and is responsible for sensory transduction. This sensory transduction channel is expressed in hair-cell stereocilia and previous studies show that its activity is affected by mutations in the genes encoding the transmembrane proteins TMHS/LHFPL5, TMIE and TMC1/2. We show here that the second ion channel is expressed at the apical surface of hair cells and contains the Piezo2 protein. The activity of the Piezo2-dependent channel is controlled by the intracellular Ca2+ concentration and can be recorded following disruption of the sensory transduction machinery or more generally by disruption of the sensory epithelium. We thus conclude that hair cells express two molecularly and functionally distinct mechanotransduction channels with different subcellular distribution.

Calcium is tightly regulated in cochlear outer hair cells (OHCs). It enters mainly via mechanotransducer (MT) channels and is extruded by the PMCA2 isoform of the plasma membrane calcium ATPase, mutations in which cause hearing loss. To assess how pump expression matches the demands of Ca2+ homeostasis, the distribution of PMCA2 at different cochlear locations during development was quantified using immunofluorescence and post-embedding immunogold labeling. The PMCA2 isoform was confined to stereociliary bundles, first appearing at the base of the cochlea around post-natal day 0 (P0) followed by the middle and then the apex by P3, and was unchanged after P8. The developmental appearance matches maturation of the MT channels in rat OHCs. High-resolution immunogold labeling in adult rats showed PMCA2 was distributed along the membranes of all three rows of OHC stereocilia at similar densities and at about a quarter the density in IHC stereocilia. The difference between OHCs and inner hair cells (IHCs) is similar to the ratio of their MT channel resting open probabilities. Gold particle counts revealed no difference in PMCA2 density between low- and high-frequency OHC bundles despite larger MT currents in high-frequency OHCs. The PMCA2 density in OHC stereocilia was determined in low- and high-frequency regions from calibration of immunogold particle counts as 2200/μm2 from which an extrusion rate of ~200 ions·s−1 per pump was inferred. The limited ability of PMCA2 to extrude the Ca2+ load through MT channels may constitute a major cause of OHC vulnerability and high-frequency hearing loss.

This composite article is intended to give the experts in the field of cochlear mechanics an opportunity to voice their personal opinion on the one mechanism they believe dominates cochlear amplification in mammals. A collection of these ideas are presented here for the auditory community and others interested in the cochlear amplifier. Each expert has given their own personal view on the topic and at the end of their commentary they have suggested several experiments that would be required for the decisive mechanism underlying the cochlear amplifier. These experiments are presently lacking but if successfully performed would have an enormous impact on our understanding of the cochlear amplifier.

Auditory discrimination is limited by the performance of the cochlea whose acute sensitivity and frequency tuning are underpinned by electromechanical feedback from the outer hair cells. Two processes may underlie this feedback: voltage-driven contractility of the outer hair cell body and active motion of the hair bundle. Either process must exert its mechanical effect via deformation of the organ of Corti, a complex assembly of sensory and supporting cells riding on the basilar membrane. Using finite element analysis, we present a three-dimensional model to illustrate deformation of the organ of Corti by the two active processes. The model used available measurements of the properties of structural components in low-frequency and high-frequency regions of the rodent cochlea. The simulations agreed well with measurements of the cochlear partition stiffness, the longitudinal space constant for point deflection, and the deformation of the organ of Corti for current injection, as well as displaying a 20-fold increase in passive resonant frequency from apex to base. The radial stiffness of the tectorial membrane attachment was found to be a crucial element in the mechanical feedback. Despite a substantial difference in the maximum force generated by hair bundle and somatic motility, the two mechanisms induced comparable amplitudes of motion of the basilar membrane but differed in the polarity of their feedback on hair bundle position. Compared to the hair bundle motor, the somatic motor was more effective in deforming the organ of Corti than in displacing the basilar membrane.

Outer hair cells (OHCs) of the mammalian cochlea besides being sensory receptors also generate force to amplify sound-induced displacements of the basilar membrane thus enhancing auditory sensitivity and frequency selectivity. This force generation is attributable to voltage-dependent contractility of the OHCs underpinned by the motile protein, prestin. Prestin is located in the basolateral wall of OHCs and is thought to alter its conformation in response to changes in membrane potential. The precise ultrastructural distribution of prestin was determined using post-embedding immunogold labelling and the density of the labelling was compared in low and high frequency regions of the cochlea. The labelling was confined to the basolateral plasma membrane in hearing rats but declined towards the base of the cells below the nucleus. In pre-hearing animals, prestin labelling was lower in the membrane and also occurred in the cytoplasm, presumably reflecting its production during development. The densities of labelling in low-frequency and high-frequency regions of the cochlea were similar. Non-linear capacitance, thought to reflect charge movements during conformational changes in prestin, was measured in OHCs in isolated cochlear coils of hearing animals. OHC non-linear capacitance in the same regions assayed in the immunolabelling was also similar in both apex and base, with charge densities of 10,000 /µm2 expressed relative to the lateral membrane area. The results suggest that prestin density and by implication force production, is similar in low-frequency and high-frequency OHCs.

This review summarizes current knowledge of the hair cell mechanotransducer channel, the ion channel responsible for detecting mechanical stimuli in the inner ear and one of the few channels whose molecular structure is still unknown. Several candidate proteins have been proposed, especially members of the transient receptor potential (TRP) channel family, but all have so far failed in one test or another. Furthermore, none has biophysical properties exactly matching the native channel. The defining features of the native mechanotransducer channel are documented, including ionic permeability, channel structure inferred from blocking agents, diversity in channel conductance, and regulation by Ca(2+), which are compared with a potential candidate, TRP channels of the polycystin family. The strengths and weaknesses of a TRP channel contender are discussed.

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.

Sound stimuli are detected in the cochlea by opening of hair cell mechanotransducer (MT) channels, one of the few ion channels not yet conclusively identified at a molecular level. To define their performancein situ, we measured MT channel properties in inner hair cells (IHCs) and outer hair cells (OHCs) at two locations in the rat cochlea tuned to different characteristic frequencies (CFs). The conductance (in 0.02 mmcalcium) of MT channels from IHCs was estimated as 260 pS at both low-frequency and mid-frequency positions, whereas that from OHCs increased with CFs from 145 to 210 pS. The combination of MT channel conductance and tip link number, assayed from scanning electron micrographs, accounts for variation in whole-cell current amplitude for OHCs and its invariance for IHCs. Channels from apical IHCs and OHCs having a twofold difference in unitary conductance were both highly calcium selective but were distinguishable by a small but significant difference in calcium permeability and in their response to lowering ionic strength. The results imply that the MT channel has properties possessed by few known candidates, and its diversity suggests expression of multiple isoforms.

There is current debate about the origin of mechanical amplification whereby outer hair cells generate force to augment the sensitivity and frequency selectivity of the mammalian cochlea. To distinguish contributions to force production from the mechanotransducer (MET) channels and somatic motility, we have measured hair bundle motion during depolarization of individual outer hair cells in isolated rat cochleas. Depolarization evoked rapid positive bundle deflections that were reduced by perfusion with the MET channel blocker dihydrostreptomycin, with no effect on the nonlinear capacitance that is a manifestation of prestin-driven somatic motility. However, the movements were also diminished by Na salicylate and depended on the intracellular anion, properties implying involvement of the prestin motor. Furthermore, depolarization of one outer hair cell caused motion of neighboring hair bundles, indicating overall motion of the reticular lamina. Depolarization of solitary outer hair cells caused cell-length changes whose voltage-activation range depended on the intracellular anion but were insensitive to dihydrostreptomycin. These results imply that both the MET channels and the somatic motor participate in hair bundle motion evoked by depolarization. It is conceivable that the two processes can interact, a signal from the MET channels being capable of modulating the activity of the prestin motor.

Cochlear hair cells respond with phenomenal speed and sensitivity to sound vibrations that cause submicron deflections of their hair bundle. Outer hair cells are not only detectors, but also generate force to augment auditory sensitivity and frequency selectivity. Two mechanisms of force production have been proposed: contractions of the cell body or active motion of the hair bundle. Here, we describe recently identified proteins involved in the sensory and motor functions of auditory hair cells and present evidence for each force generator. Both motor mechanisms are probably needed to provide the high sensitivity and frequency discrimination of the mammalian cochlea.

Calcium buffers are important for shaping and localizing cytoplasmic Ca2+transients in neurons. We measured the concentrations of the four main calcium-buffering proteins (calbindin-D28k, calretinin, parvalbumin-α, and parvalbumin-β) in rat cochlear hair cells in which Ca2+signaling is a central element of fast transduction and synaptic transmission. The proteins were quantified by calibrating immunogold tissue counts against gels containing known amounts of each protein, and the method was verified by application to Purkinje cells in which independent estimates exist for some of the protein concentrations. The results showed that, in animals with fully developed hearing, inner hair cells had\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\frac{1}{10}\) \end{document}of the proteinaceous calcium buffer of outer hair cells in which the cell body contained parvalbumin-β (oncomodulin) and calbindin-D28k at levels equivalent to 5 mmcalcium-binding sites. Both proteins were partially excluded from the hair bundles, which may permit fast unbuffered Ca2+regulation of the mechanotransducer channels. The sum of the calcium buffer concentrations decreased in inner hair cells and increased in outer hair cells as the cells developed their adult properties during cochlear maturation. The results suggest that Ca2+has distinct roles in the two types of hair cell, reflecting their different functions in auditory transduction. Ca2+is used in inner hair cells primarily for fast phase-locked synaptic transmission, whereas Ca2+may be involved in regulating the motor capability underlying cochlear amplification of the outer hair cell. The high concentration of calcium buffer in outer hair cells, similar only to skeletal muscle, may protect against deleterious consequences of Ca2+loading after acoustic overstimulation.

The narrow stimulus limits of hair cell transduction, equivalent to a total excursion of about 100 nm at the tip of the hair bundle, demand tight regulation of the mechanical input to ensure that the mechanoelectrical transducer (MET) channels operate in their linear range. This control is provided by multiple components of Ca2+–dependent adaptation. A slow mechanism limits the mechanical stimulus through the action of one or more unconventional myosins. There is also a fast, sub-millisecond, Ca2+ regulation of the MET channel, which can generate resonance and confer tuning on transduction. Changing the conductance or kinetics of the MET channels can vary their resonant frequency. The tuning information conveyed in transduction may combine with the somatic motility of outer hair cells to produce an active process that supplies amplification and augments frequency selectivity in the mammalian cochlea.

Sound stimuli vibrate the hair bundles on auditory hair cells, but the resulting motion attributable to the mechanical stimulus may be modified by forces intrinsic to the bundle, which drive it actively. One category of active hair bundle motion has properties similar to fast adaptation of the mechanotransducer channels and is explicable if gating of the channels contributes significantly to the mechanics of the hair bundle. To explore this mechanism, we measured hair bundle compliance in turtle auditory hair cells under different conditions that alter the activation range of the channel. Force–displacement relationships were nonlinear, possessing a maximum slope compliance when approximately one-half of the transducer channels were open. When the external calcium concentration was reduced from 2.8 to 0.25 mm, the position of maximum compliance was shifted negative, reflecting a comparable shift in the transducer channel activation curve. Assuming that the nonlinearity represents the compliance attributable to channel gating, a single-channel gating force of 0.25 pN was calculated. By comparing bundle displacements with depolarization with and without an attached flexible fiber, the force contributed by each channel was independently estimated as 0.47 pN. These results are consistent with fast active bundle movements resulting from changes in mechanotransducer channel gating. However, several observations revealed additional components of hair bundle motion, with slower kinetics and opposite polarity to the fast movement but also linked to transducer adaptation. This finding argues for multiple mechanisms for controlling hair bundle position in auditory hair cells.

Much is known about the mechanotransducer (MT) channels mediating transduction in hair cells of the vertrbrate inner ear. With the use of isolated preparations, it is experimentally feasible to deliver precise mechanical stimuli to individual cells and record the ensuing transducer currents. This approach has shown that small (1–100 nm) deflections of the hair-cell stereociliary bundle are transmitted via interciliary tip links to open MT channels at the tops of the stereocilia. These channels are cation-permeable with a high selectivity for Ca2+; two channels are thought to be localized at the lower end of the tip link, each with a large single-channel conductance that increases from the low- to high-frequency end of the cochlea. Ca2+influx through open channels regulates their resting open probability, which may contribute to setting the hair cell resting potential in vivo. Ca2+also controls transducer fast adaptation and force generation by the hair bundle, the two coupled processes increasing in speed from cochlear apex to base. The molecular intricacy of the stereocilary bundle and the transduction apparatus is reflected by the large number of single-gene mutations that are linked to sensorineural deafness, especially those in Usher syndrome. Studies of such mutants have led to the discovery of many of the molecules of the transduction complex, including the tip link and its attachments to the stereociliary core. However, the MT channel protein is still not firmly identified, nor is it known whether the channel is activated by force delivered through accessory proteins or by deformation of the lipid bilayer.

Sound stimuli are detected in the cochlea by vibration of hair bundles on sensory hair cells, which activates mechanotransducer ion channels and generates an electrical signal. Remarkably, the process can also work in reverse with additional force being produced by the ion channels as they open and close, evoking active movements of the hair bundle. These movements could supplement the energy of the sound stimuli but to be effective they would need to be very fast. New measurements in the turtle ear have shown that such active bundle movements occur with delays of less than a millisecond, and are triggered by the entry of Ca(2+) into the cell via the mechanotransducer channel. Furthermore, their speed depends on the frequency to which the hair cell is most sensitive, suggesting that such movements could be important in cochlear amplification and frequency discrimination.

During transduction in auditory hair cells, hair bundle deflection opens mechanotransducer channels that subsequently reclose or adapt to maintained stimuli, a major component of the adaptation occurring on a submillisecond time scale. Using a photodiode imaging technique, we measured hair bundle motion in voltage-clamped turtle hair cells to search for a mechanical correlate of fast adaptation. Excitatory force steps imposed by a flexible glass fiber attached to the bundle caused an initial movement toward the kinocilium, followed by a fast recoil equivalent to bundle stiffening. The recoil had a time course identical to adaptation of the transducer current, and like adaptation, was most prominent for small stimuli, was slowed by reducing extracellular calcium, and varied with hair cell resonant frequency. In free-standing hair bundles, depolarizations positive to 0 mV evoked an outward current attributable to opening of transducer channels, which was accompanied by a sustained bundle deflection toward the kinocilium. Both processes were sensitive to external calcium concentration and were abolished by blocking the transducer channels with dihydrostreptomycin. The similarity in properties of fast adaptation and the associated bundle motion indicates the operation of a rapid calcium-sensitive force generator linked to the gating of the transducer channels. This force generator may permit stimulus amplification during transduction in auditory hair cells.

Turtle cochlear hair cells are electrically tuned by a voltage-dependent Ca2+ current and a Ca2+-dependent K+ current (IBK(Ca)). The effects of intracellular calcium buffering on electrical tuning were studied in hair cells at apical and basal cochlear locations tuned to 100 and 300 Hz, respectively. Increasing the intracellular BAPTA concentration changed the hair cell's resonant frequency little, but optimized tuning at more depolarized membrane potentials due to a positive shift in the half-activation voltage (V½) of the IBK(Ca). The shift in V½ depended similarly on BAPTA concentration in basal and apical hair cells despite a 2·4-fold difference in the size of the Ca2+ current at the two positions. The Ca2+ current amplitude increased exponentially with distance along the cochlea. Comparison of V½ values and tuning properties using different BAPTA concentrations with values measured in perforated-patch recordings gave the endogenous calcium buffer as equivalent to 0·21 mM BAPTA in low-frequency cells, and 0·46 mM BAPTA in high-frequency cells. High conductance Ca2+-activated K+ (BKCa) channels recorded in inside-out membrane patches were 2-fold less Ca2+ sensitive in high-frequency than in low-frequency cells. Confocal Ca2+ imaging using the fluorescent indicator Calcium Green-1 revealed about twice as many hotspots of Ca2+ entry during depolarization in high-frequency compared to low-frequency hair cells. We suggest that each BKCa channel is gated by Ca2+ entry through a few nearby Ca2+ channels, and that Ca2+ and BKCa channels occupy, at constant channel density, a greater fraction of the membrane area in high-frequency cells than in low-frequency cells.

Mechanoelectrical transducer currents in turtle auditory hair cells adapted to maintained stimuli via a Ca2+-dependent mechanism characterized by two time constants of ∼1 and 15 ms. The time course of adaptation slowed as the stimulus intensity was raised because of an increased prominence of the second component. The fast component of adaptation had a similar time constant for both positive and negative displacements and was unaffected by the myosin ATPase inhibitors, vanadate and butanedione monoxime. Adaptation was modeled by a scheme in which Ca2+ions, entering through open transducer channels, bind at two intracellular sites to trigger independent processes leading to channel closure. It was assumed that the second site activates a modulator with 10-fold slower kinetics than the first site. The model was implemented by computing Ca2+diffusion within a single stereocilium, incorporating intracellular calcium buffers and extrusion via a plasma membrane CaATPase. The theoretical results reproduced several features of the experimental responses, including sensitivity to the concentration of external Ca2+and intracellular calcium buffer and a dependence on the onset speed of the stimulus. The model also generated damped oscillatory transducer responses at a frequency dependent on the rate constant for the fast adaptive process. The properties of fast adaptation make it unlikely to be mediated by a myosin motor, and we suggest that it may result from Ca2+binding to the transducer channel or a nearby cytoskeletal element.

Mechanoelectrical transducer currents in turtle auditory hair cells adapt to maintained stimuli via a Ca2+-dependent mechanism that is sensitive to the level of internal calcium buffer. We have used the properties of transducer adaptation to compare the effects of exogenous calcium buffers in the patch electrode solution with those of the endogenous buffer assayed with perforated-patch recording. The endogenous buffer of the hair bundle was equivalent to 0.1–0.4 mmBAPTA and, in a majority of cells, supported adaptation in an external Ca2+concentration of 70 μmsimilar to that in turtle endolymph. The endogenous buffer had a higher effective concentration, and the adaptation time constant was faster in cells at the high-frequency end than at the low-frequency end of the cochlea. Experiments using buffers with different Ca2+-binding rates or dissociation constants indicated that the speed of adaptation and the resting open probability of the transducer channels could be differentially regulated and imply that the endogenous buffer must be a fast, high-affinity buffer. In some hair cells, the transducer current did not decay exponentially during a sustained stimulus but displayed damped oscillations at a frequency (58–230 Hz) that depended on external Ca2+concentration. The gradient in adaptation time constant and the tuned transducer current at physiological levels of calcium buffer and external Ca2+suggest that transducer adaptation may contribute to hair cell frequency selectivity. The results are discussed in terms of feedback regulation of transducer channels mediated by Ca2+binding at two intracellular sites.

1. Recordings of mechanoelectrical transducer currents were combined with calcium imaging of hair bundles in turtle auditory hair cells located near the high-frequency end of the cochlea. The external face of the hair bundles was perfused with a range of Ca2+ concentrations to study the quantitative relationship between Ca2+ influx and transducer adaptation. 2. With Na+ as the monovalent ion, the peak amplitude of the transducer current decreased monotonically as the external [Ca2+] was raised from 25 microns to 20 mm. When Na+ was replaced with the impermeant Tris the transducer current increased with external [Ca2+]. These results indicate that Ca2+ can both permeate and block the transducer channels. The Ca2+ concentration for half-block of the monovalent current was 1 mm. 3. To quantify the Ca2+ influx, the fraction of transducer current carried by Ca2+ was measured using the change in bundle fluorescence in cells loaded with 1 mm Calcium Green-1. The fluorescence change was calibrated by substituting an impermeable monovalent ion to render Ca2+ the sole charge carrier. 4. In the presence of Na+, the fractional Ca2+ current was approximately 10% in 50 microns Ca2+, a concentration similar to that in endolymph, which bathes the hair bundles in vivo. The amount of Ca2+ entering was dependent on the identity of the monovalent ion, and was larger with K+, suggesting that the transducer channel is a multi-ion pore. 5. Over a range of ionic conditions, the rate of transducer adaptation was proportional to Ca2+ influx indicating that adaptation is driven by a rise in intracellular [Ca2+]. 6. Shifts in the current-displacement function along the displacement axis in different external Ca2+ concentrations were predictable from variation in the resting Ca2+ influx. We suggest that changes in the resting open probability of the transducer channels adjust the entry of Ca2+ to keep its concentration constant at an internal site. 7. The results demonstrate that endolymph containing high K+, 50 microns Ca2+ and low Mg2+ concentrations, maximizes the transducer current while still allowing sufficient Ca2+ entry to drive adaptation. The hair cell mechanotransducer channel, in its permeation and block by Ca2+, shows behaviour similar to the voltage-gated Ca2+ channel and the cyclic nucleotide-gated channel.

SummaryActive force generation by outer hair cells (OHCs) underlies amplification and frequency tuning in the mammalian cochlea but whether such a process exists in nonmammals is unclear. Here, we demonstrate that hair cells of the chicken auditory papilla possess an electromechanical force generator in addition to active hair bundle motion due to mechanotransducer channel gating. The properties of the force generator, its voltage dependence and susceptibility to salicylate, as well as an associated chloride-sensitive nonlinear capacitance, suggest involvement of the chicken homolog of prestin, the OHC motor protein. The presence of chicken prestin in the hair cell lateral membrane was confirmed by immunolabeling studies. The hair bundle and prestin motors together create sufficient force to produce fast lateral displacements of the tectorial membrane. Our results imply that the first use of prestin as a motor protein occurred early in amniote evolution and was not a mammalian invention as is usually supposed.

The avian auditory papilla contains two classes of sensory receptor, tall hair cells (THCs) and short hair cells (SHCs), the latter analogous to mammalian outer hair cells with large efferent but sparse afferent innervation. Little is known about the tuning, transduction, or electrical properties of SHCs. To address this problem, we made patch-clamp recordings from hair cells in an isolated chicken basilar papilla preparation at 33°C. We found that SHCs are electrically tuned by a Ca2+-activated K+ current, their resonant frequency varying along the papilla in tandem with that of the THCs, which also exhibit electrical tuning. The tonotopic map for THCs was similar to maps previously described from auditory nerve fiber measurements. SHCs also possess an A-type K+ current, but electrical tuning was observed only at resting potentials positive to −45 mV, where the A current is inactivated. We predict that the resting potential in vivo is approximately −40 mV, depolarized by a standing inward current through mechanotransducer (MT) channels having a resting open probability of ∼0.26. The resting open probability stems from a low endolymphatic Ca2+ concentration (0.24 mM) and a high intracellular mobile Ca2+ buffer concentration, estimated from perforated-patch recordings as equivalent to 0.5 mM BAPTA. The high buffer concentration was confirmed by quantifying parvalbumin-3 and calbindin D-28K with calibrated postembedding immunogold labeling, demonstrating >1 mM calcium-binding sites. Both proteins displayed an apex-to-base gradient matching that in the MT current amplitude, which increased exponentially along the papilla. Stereociliary bundles also labeled heavily with antibodies against the Ca2+ pump isoform PMCA2a.

Confocal imaging has revealed microdomains of intracellular free Ca2+ in turtle hair cells evoked by depolarizing pulses and has delineated factors affecting the growth and dissipation of such domains. However, imaging experiments have limited spatial and temporal resolution. To extend the range of the results we have developed a three-dimensional model of Ca2+ diffusion in a cylindrical hair cell, allowing part of the Ca2+ influx to occur over a small circular region (radius 0.125–1.0 micron) representing a high-density array of voltage-dependent channels. The model incorporated experimental information about the number of channels, the fixed and mobile Ca2+ buffers, and the Ca2+ extrusion mechanism. A feature of the calculations was the use of a variable grid size depending on the proximity to the Ca2+ channel cluster. The results agreed qualitatively with experimental data on the localization of the Ca2+ transients, although the experimental responses were smaller and slower, which is most likely due to temporal and spatial averaging in the imaging. The model made predictions about 1) the optimal Ca2+ channel number and density within a cluster, 2) the conditions to ensure independence of neighboring clusters, and 3) the influence of the Ca2+ buffers on the kinetics and localization of the microdomains. We suggest that an increase in the mobile Ca2+ buffer concentration in high-frequency hair cells (which possess a larger number of release sites) would allow lower amplitude and faster Ca2+ responses and promote functional independence of the sites.

Hair cells in the turtle cochlea are frequency-tuned by a mechanism involving the combined activation of voltage-sensitive Ca2+ channels and Ca(2+)-activated K+ (KCa) channels. The main determinants of a hair cell's characteristic frequency (Fo) are the KCa channels' density and kinetics, both of which change systematically with location in the cochlea in conjunction with the observed frequency map. We have developed a model based on the differential expression of two KCa channel subunits, which when accompanied by concurrent changes in other properties (e.g., density of Ca2+ channels and inwardly rectifying K+ channels), will generate sharp tuning at frequencies from 40 to 600 Hz. The kinetic properties of the two subunits were derived from previous single-channel analysis, and it was assumed that the subunits (A and B) combine to form five species of tetrameric channel (A4, A3B, A2B2, AB3, and B4) with intermediate kinetics and overlapping distribution. Expression of KCa and other channels was assumed to be regulated by diffusional gradients in either one or two chemicals. The results are consistent with both current- and voltage-clamp data on turtle hair cells, and they show that five channel species are sufficient to produce smooth changes in both Fo and kinetics of the macroscopic KCa current. Other schemes for varying KCa channel kinetics are examined, including one that allows extension of the model to the chick cochlea to produce hair cells with Fo's from 130 to 4000 Hz. A necessary assumption in all models is a gradient in the values of the parameters identified with the cell's cytoplasmic Ca2+ buffer.

A major factor determining the electrical resonant frequency of turtle cochlear hair cells is the time course of the Ca-activated K current (Art, J. J., and R. Fettiplace. 1987. Journal of Physiology. 385:207-242). We have examined the notion that this time course is dictated by the K channel kinetics by recording single Ca-activated K channels in inside-out patches from isolated cells. A hair cell's resonant frequency was estimated from its known correlation with the dimensions of the hair bundle. All cells possess BK channels with a similar unit conductance of approximately 320 pS but with different mean open times of 0.25-12 ms. The time constant of relaxation of the average single-channel current at -50 mV in 4 microM Ca varied between cells from 0.4 to 13 ms and was correlated with the hair bundle height. The magnitude and voltage dependence of the time constant agree with the expected behavior of the macroscopic K(Ca) current, whose speed may thus be limited by the channel kinetics. All BK channels had similar sensitivities to Ca which produced half-maximal activation for a concentration of approximately 2 microM at +50 mV and 12 microM at -50 mV. We estimate from the voltage dependence of the whole-cell K(Ca) current that the BK channels may be fully activated at -35 mV by a rise in intracellular Ca to 50 microM. BK channels were occasionally observed to switch between slow and fast gating modes which raises the possibility that the range of kinetics of BK channels observed in different hair cells reflects a common channel protein whose kinetics are regulated by an unidentified intracellular factor. Membrane patches also contained 30 pS SK channels which were approximately 5 times more Ca-sensitive than BK channels at -50 mV. The SK channels may underlie the inhibitory synaptic potential produced in hair cells by efferent stimulation.

Two types of mechanosensitive (MS) currents have been described in cochlear hair cells: conventional MS currents evoked by displacements of the hair bundle towards its tallest edge, and anomalous MS currents, elicited by bundle displacements in the opposite direction, referred to as reversed-polarity currents. Conventional MS transducer channels are located at the bottom end of the tip links, but reverse-polarity MS currents persist after severing tip links with BAPTA, suggesting a different site for this channel. We investigated localization of reverse-polarity MS channels using cell-attached patch recordings on cochlear outer hair cells (OHC) of neonatal mice, and also by detecting calcium influx with Fluo5F indicator using swept-field confocal imaging. Single mechano-sensitive channels were recorded in cell-attached mode on the apical surface around the base of the hair bundle and were activated by suction through the patch pipette. In OHCs of wild-type mice, MS channels appeared a few minutes after BAPTA exposure with a mean conductance of 61 ± 6 pS in Na-saline with 1.5 mM CaCl2 and 94 ± 13 pS in Na-saline with reduced, 0.07 mM, CaCl2. Their reversal potential was 0 mV as expected for a non-selective cationic channel. Similar MS channels were also seen in Tmc1:Tmc2 double mutants, with mean conductance of 58 ± 4 pS in 1.5 mM CaCl2. In both cases, the apical surface of the OHC was confirmed as the main site of calcium entry during reverse polarity MS currents by imaging calcium influx. The properties of the reverse-polarity MS channels closely resemble conventional MS channels but they are concentrated on the apical plasma membrane of the hair cell. We suggest they reflect insertion of new MS channels before their transport up the bundle to the transduction complex.

Spontaneous Ca(2+)-dependent electrical activity in the immature mammalian cochlea is thought to instruct the formation of the tonotopic map during the differentiation of sensory hair cells and the auditory pathway. This activity occurs in inner hair cells (IHCs) during the first postnatal week, and the pattern differs along the cochlea. During the second postnatal week, which is before the onset of hearing in most rodents, the resting membrane potential for IHCs is apparently more hyperpolarized (approximately -75 mV), and it remains unclear whether spontaneous action potentials continue to occur. We found that when mouse IHC hair bundles were exposed to the estimated in vivo endolymphatic Ca(2+) concentration (0.3 mm) present in the immature cochlea, the increased open probability of the mechanotransducer channels caused the cells to depolarize to around the action potential threshold (approximately -55 mV). We propose that, in vivo, spontaneous Ca(2+) action potentials are intrinsically generated by IHCs up to the onset of hearing and that they are likely to influence the final sensory-independent refinement of the developing cochlea.