Laura F. Corns

Calcium entry into stereocilia drives adaptation of the mechanoelectrical transducer current of mammalian cochlear hair cells

Significance In the inner ear, the sensory receptor cells (hair cells) signal reception of sound. They do so by converting mechanical input, due to sound waves moving the hair bundles on these cells, into electrical current through ion channels situated at the tips of the bundles. To keep the receptors operating at their maximum sensitivity, the current declines rapidly, a process known as adaptation. In nonmammalian vertebrates, Ca2+ ions entering the mechanosensitive ion channels drive adaptation, but it has been questioned whether this mechanism applies to mammals. We show that adaptation in mammalian cochlear hair cells is, as in other vertebrates, driven by Ca2+ entry, demonstrating the importance of this process as a fundamental mechanism in vertebrate hair cells. 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, Ca2+ entry through the open MET channels causes adaptation, rapidly reducing their open probability and resetting their operating range. It remains uncertain whether such Ca2+-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 Ca2+ reversal potential or their hair bundles were exposed to the in vivo endolymphatic Ca2+ 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 Ca2+ 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 Ca2+, intracellular Ca2+ buffering, and membrane potential, by their common effect on intracellular free Ca2+.

Transduction without Tip Links in Cochlear Hair Cells Is Mediated by Ion Channels with Permeation Properties Distinct from Those of the Mechano-Electrical Transducer Channel

Tip links between adjacent stereocilia are believed to gate mechano-electrical transducer (MET) channels and mediate the electrical responses of sensory hair cells. We found that mouse auditory hair cells that lack tip links due to genetic mutations or exposure to the Ca2+ 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.

Tmc1 point mutation affects Ca2+ sensitivity and block by dihydrostreptomycin of the mechanoelectrical transducer current of mouse outer hair cells

The transduction of sound into electrical signals depends on mechanically sensitive ion channels in the stereociliary bundle. The molecular composition of this mechanoelectrical transducer (MET) channel is not yet known. Transmembrane channel-like protein isoforms 1 (TMC1) and 2 (TMC2) have been proposed to form part of the MET channel, although their exact roles are still unclear. Using Beethoven (Tmc1Bth/Bth) mice, which have an M412K point mutation in TMC1 that adds a positive charge, we found that Ca2+ permeability and conductance of the MET channel of outer hair cells (OHCs) were reduced. Tmc1Bth/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 Ca2+ 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 Tmc1Bth/Bth OHCs, whereas raising the intracellular concentration of the Ca2+ 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 Ca2+ permeability of the mutated MET channel indirectly causing the Ca2+ sensor for adaptation, at or near the intracellular face of the MET channel, to become more sensitive to Ca2+ influx as a compensatory mechanism. SIGNIFICANCE STATEMENT In the auditory system, the hair cells convert sound-induced mechanical movement of the hair bundles atop these cells into electrical signals through the opening of mechanically gated ion channels at the tips of the bundles. Although the nature of these mechanoelectrical transducer (MET) channels is still unclear, recent studies implicate transmembrane channel-like protein isoform 1 (TMC1) channels in the mammalian cochlea. Using a mutant mouse model (Beethoven) for progressive hearing loss in humans (DFNA36), which harbors a point mutation in the Tmc1 gene, we show that this mutation affects the MET channel pore, reducing its Ca2+ permeability and its affinity for the permeant blocker dihydrostreptomycin. A number of phenomena that we ascribe to Ca2+-dependent adaptation appear stronger, in compensation for the reduced Ca2+ entry.

The acquisition of mechano‐electrical transducer current adaptation in auditory hair cells requires myosin VI

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 (Snells 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.

GABAergic responses of mammalian ependymal cells in the central canal neurogenic niche of the postnatal spinal cord.

Highlights • Extensive dye-coupling occurs between mammalian spinal cord ependymal cells.• GABA depolarised all spinal cord ependymal cells tested.• GABA effects were mediated by GABAA receptors but not GABA uptake transporters.

Kv3.3 immunoreactivity in the vestibular nuclear complex of the rat with focus on the medial vestibular nucleus: Targeting of Kv3.3 neurones by terminals positive for vesicular glutamate transporter 1

Kv3 voltage-gated K(+) channels are important in shaping neuronal excitability and are abundant in the CNS, with each Kv3 gene exhibiting a unique expression pattern. Mice lacking the gene encoding for the Kv3.3 subunit exhibit motor deficits. Furthermore, mutations in this gene have been linked to the human disease spinocerebellar ataxia 13, associated with cerebellar and extra-cerebellar symptoms such as imbalance and nystagmus. Kv subunit localisation is important in defining their functional roles and thus, we investigated the distribution of Kv3.3-immunoreactivity in the vestibular nuclear complex of rats with particular focus on the medial vestibular nucleus (MVN). Kv3.3-immunoreactivity was widespread in the vestibular nuclei and was detected in somata, dendrites and synaptic terminals. Kv3.3-immunoreactivity was observed in distinct neuronal populations and dual labelling with the neuronal marker NeuN revealed 28.5+/-1.9% of NeuN labelled MVN neurones were Kv3.3-positive. Kv3.3-immunoreactivity co-localised presynaptically with the synaptic vesicle marker SV2, parvalbumin, the vesicular glutamate transporter VGluT2 and the glycine transporter GlyT2. VGluT1 terminals were scarce within the MVN (2.5+/-1.1 per 50 microm(2)) and co-localisation was not observed. However, 85.4+/-9.4% of VGluT1 terminals targeted and enclosed Kv3.3-immunoreactive somata. Presynaptic Kv3.3 co-localisation with the GABAergic marker GAD67 was also not observed. Cytoplasmic GlyT2 labelling was observed in a subset of Kv3.3-positive neurones. Electron microscopy confirmed a pre- and post-synaptic distribution of the Kv3.3 protein. This study provides evidence supporting a role for Kv3.3 subunits in vestibular processing by regulating neuronal excitability pre- and post-synaptically.

TMC2 Modifies Permeation Properties of the Mechanoelectrical Transducer Channel in Early Postnatal Mouse Cochlear Outer Hair Cells

The ability of cochlear hair cells to convert sound into receptor potentials relies on the mechanoelectrical transducer (MET) channels present in their stereociliary bundles. There is strong evidence implying that transmembrane channel-like protein (TMC) 1 contributes to the pore-forming subunit of the mature MET channel, yet its expression is delayed (~>P5 in apical outer hair cells, OHCs) compared to the onset of mechanotransduction (~P1). Instead, the temporal expression of TMC2 coincides with this onset, indicating that it could be part of the immature MET channel. We investigated MET channel properties from OHCs of homo- and heterozygous Tmc2 knockout mice. In the presence of TMC2, the MET channel blocker dihydrostreptomycin (DHS) had a lower affinity for the channel, when the aminoglycoside was applied extracellularly or intracellularly, with the latter effect being more pronounced. In Tmc2 knockout mice OHCs were protected from aminoglycoside ototoxicity during the first postnatal week, most likely due to their small MET current and the lower saturation level for aminoglycoside entry into the individual MET channels. DHS entry through the MET channels of Tmc2 knockout OHCs was lower during the first than in the second postnatal week, suggestive of a developmental change in the channel pore properties independent of TMC2. However, the ability of TMC2 to modify the MET channel properties strongly suggests it contributes to the pore-forming subunit of the neonatal channel. Nevertheless, we found that TMC2, different from TMC1, is not necessary for OHC development. While TMC2 is required for mechanotransduction in mature vestibular hair cells, its expression in the immature cochlea may be an evolutionary remnant.

Piezo1 haploinsufficiency does not alter mechanotransduction in mouse cochlear outer hair cells.

The mechanoelectrical transducer (MET) channels located at the stereocilia tip of cochlear hair cells are crucial to convert the mechanical energy of sound into receptor potentials, but the identity of its pore‐forming subunits remains uncertain. Piezo1, which has been identified in the transcriptome of mammalian cochlear hair cells, encodes a transmembrane protein that forms mechanosensitive channels in other tissues. We investigated the properties of the MET channel in outer hair cells (OHCs) of Piezo1 mice (postnatal day 6–9). The MET current was elicited by deflecting the hair bundle of OHCs using sinewave and step stimuli from a piezo‐driven fluid jet. Apical and basal OHCs were investigated because the properties of the MET channel vary along the cochlea. We found that the maximal MET current amplitude and the resting open probability of the MET channel in OHCs were similar between Piezo1+/− haploinsufficient mice and wild‐type littermates. The sensitivity to block by the permeant MET channel blocker dihydrostreptomycin was also similar between the two genotypes. Finally, the anomalous mechano‐gated current, which is activated by sheer force and which is tip‐link independent, was unaffected in OHCs from Piezo1+/− haploinsufficient mice. Our results suggest that Piezo1 is unlikely to be a component of the MET channel complex in mammalian cochlear OHCs.

Mechanotransduction is required for establishing and maintaining mature inner hair cells and regulating efferent innervation

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.Mechanoelectrical transducer (MET) channels on the tips of inner hair cells are essential for transducing auditory sensory information. Here, the authors show that disrupting MET channel function also prevents the preservation of normal inner hair cell identity in adult mice.