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

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

Cochlear tonotopy from proteins to perception.

A ubiquitous feature of the auditory organ in amniotes is the longitudinal mapping of neuronal characteristic frequencies (CFs), which increase exponentially with distance along the organ. The exponential tonotopic map reflects variation in hair cell properties according to cochlear location and is thought to stem from concentration gradients in diffusible morphogenic proteins during embryonic development. While in all amniotes the spatial gradient is initiated by sonic hedgehog (SHH), released from the notochord and floorplate, subsequent molecular pathways are not fully understood. In chickens, BMP7 is one such morphogen, secreted from the distal end of the cochlea. In mammals, the developmental mechanism differs from birds and may depend on cochlear location. A consequence of exponential maps is that each octave occupies an equal distance on the cochlea, a spacing preserved in the tonotopic maps in higher auditory brain regions. This may facilitate frequency analysis and recognition of acoustic sequences.

The conductance and organization of the TMC1-containing mechanotransducer channel complex in auditory hair cells.

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 Ca2+ 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 Ca2+ 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.

Atypical tuning and amplification mechanisms in gecko auditory hair cells.

SignificanceGeckos are lizards capable of vocalization and can detect frequencies up to 5 kHz, but the mechanism of frequency discrimination is incompletely understood. The gecko's auditory papilla has a unique arrangement over the high-frequency zone, with rows of mechanically sensitive hair bundles covered with gelatinous sallets. Lower-frequency hair cells are tuned by an electrical resonance employing Ca2+-activated K+ channels, but hair cells tuned above 1 kHz probably rely on a mechanical resonance of the sallets. The resonance may be boosted by an electromotile force from hair bundles found to be evoked by changes in hair cell membrane potential. This unusual mechanism operates independently of mechanotransduction and differs from mammals which amplify the mechanical input using the motor protein prestin.

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

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

New Tmc1 Deafness Mutations Impact Mechanotransduction in Auditory Hair Cells.

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, TMC1 p.T422K, and have characterized the homologous mouse mutant, Tmc1 p.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 are TMC1 p.L416R, p.G417R, and p.M418K, the last homologous to the mouse Beethoven that 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 (Tmc1 p.T416K, Tmc1 p.D569N) and recessive (Tmc1 p.W554L, Tmc1 p.D528N) mutations of residues near the putative pore of the channel. Tmc1 p.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. Tmc1 p.D528N, located in the pore's narrowest region, uniquely caused large reductions in MET channel conductance and block by dihydrostreptomycin (DHS). For Tmc1 p.T416K and Tmc1 p.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 STATEMENT Transmembrane 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 harboring Tmc1 point 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.

Diverse Mechanisms of Sound Frequency Discrimination in the Vertebrate Cochlea.

Discrimination of different sound frequencies is pivotal to recognizing and localizing friend and foe. Here, I review the various hair cell-tuning mechanisms used 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 ~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 uses somatic motility of outer hair cells, underpinned by the membrane protein prestin, to expand the frequency range. The bird cochlea may also use prestin at high frequencies, but hair cells <1 kHz show electrical resonance.

The contribution of TMC1 to adaptation of mechanoelectrical transduction channels in cochlear outer 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. 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.

A Tmc1 mutation reduces calcium permeability and expression of mechanoelectrical transduction channels in cochlear hair cells.

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.

Tonotopy in calcium homeostasis and vulnerability of cochlear hair cells.

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 Ca2+ balance among cochlear locations. Homeostasis is determined by three factors: Ca2+ 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 Ca2+ during Ca2+ influx through the MET channels. We suggest that, in OHCs, there are two distinct micro-compartments for Ca2+ 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 Ca2+ 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.

Variable number of TMC1-dependent mechanotransducer channels underlie tonotopic conductance gradients in the cochlea.

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.

Spatiotemporal changes in the distribution of LHFPL5 in mice cochlear hair bundles during development and in the absence of PCDH15.

Mechanosensory transduction by vertebrate hair cells depends on a protein complex at the tips of shorter stereocilia associated with mechanoelectrical transduction channels activated by tip links in the hair bundle. In mammalian hair cells, this complex includes transmembrane channel-like protein subunit 1 (TMC1), lipoma HMGIC fusion partner-like 5 protein (LHFPL5) and protocadherin 15 (PCDH15), a lower-end component of the tip link. TMC1 interacts with LHFPL5 and PCDH15 but how the complex develops to maturity, and the relationships between these proteins, remains uncertain. Here we evaluate the spatiotemporal development of LHFPL5 distributions in mouse cochlear hair bundles by immunofluorescence and immunogold transmission electron microscopy, from postnatal day 0 (P0) through P21 in wild type and PCDH15-deficient mice. At P0, hair bundles contain many short microvilli-like processes which we term unranked stereocilia, and a subset of lengthening rows, adjacent to a kinocilium. LHFPL5 is distributed throughout the bundle, including on stereocilia tips and the kinocilium. At P3, 4-to-6 rows of ranked stereocilia are evident, total LHFPL5 expression peaks, and LHFPL5 is localised to ranked stereocilia tips of all rows and to lower shaft/ankle links. By P12, the bundle has a mature pattern with 3 ranked rows but virtually no unranked stereocilia or kinocilium; LHFPL5 expression has declined and become restricted to the tips of shorter stereocilia. Throughout development from P0, expression of LHFPL5 is greater overall on apical than basal bundles, but there is, on average, an equal amount of labelling per labelled tip. In P3 mice lacking PCDH15, LHFPL5 labelling is not at the tips but is primarily on unranked stereocilia and lower lateral links. These data show that LHFPL5 is already present in the MET apparatus at P0 but requires PCDH15 at P3 to remain there. Shaft/ankle link localisation suggests it interacts with link proteins other than PCDH15.

PIEZO2 as the anomalous mechanotransducer channel in auditory hair cells.

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.

Hair Cell Transduction, Tuning, and Synaptic Transmission in the Mammalian Cochlea.

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.

CIB2 interacts with TMC1 and TMC2 and is essential for mechanotransduction in auditory hair cells.

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.

Mechanosensory hair cells express two molecularly distinct mechanotransduction channels.

Auditory hair cells contain mechanotransduction channels that rapidly open in response to sound-induced vibrations. 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, TMIE, TMC1 and TMC2. We show here that the second ion channel is expressed at the apical surface of hair cells and that it 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 distributions.

Evaluation of Nestin Expression in the Developing and Adult Mouse Inner Ear.

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.

Is TMC1 the Hair Cell Mechanotransducer Channel?

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 Ca(2+) 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.

Development and localization of reverse-polarity mechanotransducer channels in cochlear hair cells.

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.

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 Ca(2+). The Ca(2+)-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 Ca(2+); these effects may be attributed to the channel's reduced Ca(2+) permeability. Moreover, the density of stereociliary CaATPase pumps for Ca(2+) extrusion was decreased in the mutant. The results suggest that a major component of channel adaptation is regulated by changes in intracellular Ca(2+). Consistent with this idea, the adaptive shift in the current-displacement relationship when hair bundles were bathed in endolymph-like Ca(2+) saline was usually abolished by raising the intracellular Ca(2+) concentration.