Mechanotransduction in the auditory and vestibular systems depends on mechanosensitive ion channels in the stereociliary bundles that project from the apical surface of the sensory hair cells. In lower vertebrates, when the mechanoelectrical transducer (MET) channels are opened by movement of the bundle in the excitatory direction, Ca 2+ entry through the open MET channels causes adaptation, rapidly reducing their open probability and resetting their operating range. It remains uncertain whether such Ca 2+ -dependent adaptation is also present in mammalian hair cells. Hair bundles of both outer and inner hair cells from mice were deflected by using sinewave or step mechanical stimuli applied using a piezo-driven fluid jet. We found that when cochlear hair cells were depolarized near the Ca 2+ reversal potential or their hair bundles were exposed to the in vivo endolymphatic Ca 2+ concentration (40 μM), all manifestations of adaptation, including the rapid decline of the MET current and the reduction of the available resting MET current, were abolished. MET channel adaptation was also reduced or removed when the intracellular Ca 2+ buffer 1,2-Bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) was increased from a concentration of 0.1 to 10 mM. The findings show that MET current adaptation in mouse auditory hair cells is modulated similarly by extracellular Ca 2+ , intracellular Ca 2+ buffering, and membrane potential, by their common effect on intracellular free Ca 2+ .H earing and balance depend on the transduction of mechanical stimuli into electrical signals. This process depends on the opening of mechanoelectrical transducer (MET) channels located at the tips of the shorter of pairs of adjacent stereocilia (1), which are specialized microvilli-like structures that form the hair bundles that project from the upper surface of hair cells (2,3). Deflection of hair bundles in the excitatory direction (i.e., toward the taller stereocilia) stretches specialized linkages, the tip-links, present between adjacent stereocilia (3-5), opening the MET channels. In hair cells from lower vertebrates, open MET channels reclose during constant stimuli via an initial fast adaptation mechanism followed by a much slower, myosin-based motor process, both of which are driven by Ca 2+ entry through the channel itself (6-13). In mammalian auditory hair cells, MET current adaptation seems to be mainly driven by the fast mechanism (14-16), although the exact process by which it occurs is still largely unknown. The submillisecond speed associated with the adaptation kinetics of the MET channels in rat and mouse cochlear hair cells (17,18) indicates that Ca 2+ , to cause adaptation, has to interact directly with a binding site on the channel or via an accessory protein (16). However, a recent investigation on rat auditory hair cells has challenged the view that Ca 2+ entry is required for fast adaptation, and instead proposed an as-yetundefined mechanism involving a Ca 2+ -independent reduction in the viscoelastic force of ele...

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

Bth/Bth OHCs were also less sensitive to block by the permeant MET channel blocker dihydrostreptomycin, whether applied extracellularly or intracellularly. These findings suggest that the amino acid that is mutated in Bth is situated at or near the negatively charged binding site for dihydrostreptomycin within the permeation pore of the channel. We also found that the Ca 2ϩ dependence of the operating range of the MET channel was altered by the M412K mutation. Depolarization did not increase the resting open probability of the MET current of Tmc1 Bth/Bth OHCs, whereas raising the intracellular concentration of the Ca 2ϩ chelator BAPTA caused smaller increases in resting open probability in Bth mutant OHCs than in wild-type control cells. We propose that these observations can be explained by the reduced Ca 2ϩ permeability of the mutated MET channel indirectly causing the Ca 2ϩ sensor for adaptation, at or near the intracellular face of the MET channel, to become more sensitive to Ca 2ϩ influx as a compensatory mechanism.

In the adult auditory organ, mechanoelectrical transducer (MET) channels are essential for transducing acoustic stimuli into electrical signals. In the absence of incoming sound, a fraction of the MET channels on top of the sensory hair cells are open, resulting in a sustained depolarizing current. By genetically manipulating the in vivo expression of molecular components of the MET apparatus, we show that during pre-hearing stages the MET current is essential for establishing the electrophysiological properties of mature inner hair cells (IHCs). If the MET current is abolished in adult IHCs, they revert into cells showing electrical and morphological features characteristic of pre-hearing IHCs, including the re-establishment of cholinergic efferent innervation. The MET current is thus critical for the maintenance of the functional properties of adult IHCs, implying a degree of plasticity in the mature auditory system in response to the absence of normal transduction of acoustic signals.

Key points The transduction of sound into electrical signals occurs at the hair bundles atop sensory hair cells in the cochlea, by means of mechanosensitive ion channels, the mechano‐electrical transducer (MET) channels.The MET currents decline during steady stimuli; this is termed adaptation and ensures they always work within the most sensitive part of their operating range, responding best to rapidly changing (sound) stimuli.In this study we used a mouse model (Snell's waltzer) for hereditary deafness in humans that has a mutation in the gene encoding an unconventional myosin, myosin VI, which is present in the hair bundles.We found that in the absence of myosin VI the MET current fails to acquire its characteristic adaptation as the hair bundles develop.We propose that myosin VI supports the acquisition of adaptation by removing key molecules from the hair bundle that serve a temporary, developmental role. AbstractMutations in Myo6, the gene encoding the (F‐actin) minus end‐directed unconventional myosin, myosin VI, cause hereditary deafness in mice (Snell's waltzer) and humans. In the sensory hair cells of the cochlea, myosin VI is expressed in the cell bodies and along the stereocilia that project from the cells’ apical surface. It is required for maintaining the structural integrity of the mechanosensitive hair bundles formed by the stereocilia. In this study we investigate whether myosin VI contributes to mechano‐electrical transduction. We report that Ca2+‐dependent adaptation of the mechano‐electrical transducer (MET) current, which serves to keep the transduction apparatus operating within its most sensitive range, is absent in outer and inner hair cells from homozygous Snell's waltzer mutant mice, which fail to express myosin VI. The operating range of the MET channels is also abnormal in the mutants, resulting in the absence of a resting MET current. We found that cadherin 23, a component of the hair bundle's transient lateral links, fails to be downregulated along the length of the stereocilia in maturing Myo6 mutant mice. MET currents of heterozygous littermates appear normal. We propose that myosin VI, by removing key molecules from developing hair bundles, is required for the development of the MET apparatus and its Ca2+‐dependent adaptation.