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.
Myosin-VIIA (MYO7A) is an unconventional myosin responsible for syndromic (Usher 1B) or nonsyndromic forms of deafness in humans when mutated. In the cochlea, MYO7A is expressed in hair cells, where it is believed to act as the motor protein tensioning the mechanoelectrical transducer (MET) channels, thus setting their resting open probability ( P o ). However, direct evidence for this unique role for an unconventional myosin in mature hair cells is lacking. Here, we show that MYO7A has a distinct role in hair cells, being crucial for the structural integrity of hair bundles. Postnatal deletion of Myo7a leads to 87 to 96% reduction in MYO7A from hair cells by postnatal day 20 (P20), without affecting hearing function. During the following week, mice showed progressive decline in both hearing function and MET current amplitude in hair cells without affecting the resting P o and calcium sensitivity of the MET channel. Hair-bundle stiffness was normal at P20 but halved at P30, despite it having a normal staircase morphology and tip links. The reduction of MYO7A in the stereocilia (>87%) increased their vulnerability to sound-induced damage, with significantly more hearing loss and hair bundle deterioration than in control mice. RNA-sequencing identified a downregulation of several stereociliary genes in the Myo7a -deficient cochlea, indicating the presence of indirect compensatory mechanisms. This study reveals that mature hair cells seem to use a MYO7A-independent mechanism to maintain the resting P o of the MET channels. Instead, MYO7A is essential for maintaining the structural and functional integrity of the hair bundles.
Mutations in the genes encoding for gap junction proteins connexin 26 (Cx26) and connexin 30 (Cx30) have been linked to syndromic and nonsyndromic hearing loss in mice and humans. The release of ATP from connexin hemichannels in cochlear nonsensory cells has been proposed to be the main trigger for action potential activity in immature sensory inner hair cells (IHCs), which is crucial for the refinement of the developing auditory circuitry. Using connexin knock-out mice, we show that IHCs fire spontaneous action potentials even in the absence of ATP-dependent intercellular Ca2+ signaling in the nonsensory cells. However, this signaling from nonsensory cells was able to increase the intrinsic IHC firing frequency. We also found that connexin expression is key to IHC functional maturation. In Cx26 conditional knock-out mice (Cx26Sox10-Cre), the maturation of IHCs, which normally occurs at approximately postnatal day 12, was partially prevented. Although Cx30 has been shown not to be required for hearing in young adult mice, IHCs from Cx30 knock-out mice exhibited a comprehensive brake in their development, such that their basolateral membrane currents and synaptic machinery retain a prehearing phenotype. We propose that IHC functional differentiation into mature sensory receptors is initiated in the prehearing cochlea provided that the expression of either connexin reaches a threshold level. As such, connexins regulate one of the most crucial functional refinements in the mammalian cochlea, the disruption of which contributes to the deafness phenotype observed in mice and DFNB1 patients. SIGNIFICANCE STATEMENT The correct development and function of the mammalian cochlea relies not only on the sensory hair cells, but also on the surrounding nonsensory cells. Although the nonsensory cells have been largely implicated in the general homeostasis in the mature cochlea, their involvement in the initial functional differentiation of the sensory inner hair cells is less clear. Using mutant mouse models for the most common form of congenital deafness in humans, which are knock-outs for the gap-junction channels connexin 26 and connexin 30 genes, we show that defects in nonsensory cells prevented the functional maturation of inner hair cells. In connexin knock-outs, inner hair cells remained stuck at a prehearing stage of development and, as such, are unable to process sound information.
The maintenance of balance and gaze relies on the faithful and rapid signaling of head movements to the brain. In mammals, vestibular organs contain two types of sensory hair cells, type-I and type-II, which convert the head motion-induced movement of their hair bundles into a graded receptor potential that drives action potential activity in their afferent fibers. While signal transmission in both hair cell types involves Ca2+-dependent quantal release of glutamate at ribbon synapses, type-I cells appear to also exhibit a non-quantal mechanism that is believed to increase transmission speed. However, the reliance of mature type-I hair cells on non-quantal transmission remains unknown. Here we investigated synaptic transmission in mammalian utricular hair cells using patch-clamp recording of Ca2+ currents and changes in membrane capacitance (ΔCm). We found that mature type-II hair cells showed robust exocytosis with a high-order dependence on Ca2+ entry. By contrast, exocytosis was approximately 10 times smaller in type-I hair cells. Synaptic vesicle exocytosis was largely absent in mature vestibular hair cells of CaV1.3 (CaV1.3-/- ) and otoferlin (Otof-/- ) knockout mice. Even though Ca2+-dependent exocytosis was small in type-I hair cells of wild-type mice, or absent in CaV1.3-/- and Otof-/- mice, these cells were able to drive action potential activity in the postsynaptic calyces. This supports a functional role for non-quantal synaptic transmission in type-I cells. The large vesicle pools in type-II cells would facilitate sustained transmission of tonic or low-frequency signals. In type-I cells, the restricted vesicle pool size, together with a rapid non-quantal mechanism, could allow them to sustain high-frequency phasic signal transmission at their specialized large calyceal synapses.
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 ( Tmc1 Bth/Bth ) mice, which have an M412K point mutation in TMC1 that adds a positive charge, we found that Ca 2+ permeability and conductance of the MET channel of outer hair cells (OHCs) were reduced. Tmc1 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 2+ 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 2+ 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 2+ permeability of the mutated MET channel indirectly causing the Ca 2+ sensor for adaptation, at or near the intracellular face of the MET channel, to become more sensitive to Ca 2+ 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 Ca 2+ permeability and its affinity for the permeant blocker dihydrostreptomycin. A number of phenomena that we ascribe to Ca 2+ -dependent adaptation appear stronger, in compensation for the reduced Ca 2+ entry.
Age-related hearing loss (ARHL) is associated with the loss of inner hair cell (IHC) ribbon synapses, lower hearing sensitivity and decreased ability to understand speech, especially in a noisy environment. Little is known about the age-related physiological and morphological changes that occur at ribbon synapses. We show that the differing degrees of ARHL in four selected mouse stains is correlated with the loss of ribbon synapses, being most severe for the strains C57BL/6NTac and C57BL/6J, less so for C57BL/6NTacCdh23+ -Repaired and lowest for C3H/HeJ. Despite the loss of ribbon synapses with age, the volume of the remaining ribbons increased and the size and kinetics of Ca2+ -dependent exocytosis in IHCs was unaffected, indicating the presence of a previously unknown degree of functional compensation at ribbon synapses. Although the age-related morphological changes at IHC ribbon synapses contribute to the different progression of ARHL, without the observed functional compensation hearing loss could be greater.Mammalian cochlear inner hair cells (IHCs) are specialized sensory receptors able to provide dynamic coding of sound signals. This ability is largely conferred by their ribbon synapses, which tether a large number of vesicles at the IHC's presynaptic active zones, allowing high rates of sustained synaptic transmission onto the afferent fibres. How the physiological and morphological properties of ribbon synapses change with age remains largely unknown. Here, we have investigated the biophysical and morphological properties of IHC ribbon synapses in the ageing cochlea (9-12 kHz region) of four mouse strains commonly used in hearing research: early-onset progressive hearing loss (C57BL/6J and C57BL/6NTac) and 'good hearing' strains (C57BL/6NTacCdh23+ and C3H/HeJ). We found that with age, both modiolar and pillar sides of the IHC exhibited a loss of ribbons, but there was an increased volume of those that remained. These morphological changes, which only occurred after 6 months of age, were correlated with the level of hearing loss in the different mouse strains, being most severe for C57BL/6NTac and C57BL/6J, less so for C57BL/6NTacCdh23+ and absent for C3H/HeJ strains. Despite the age-related reduction in ribbon number in three of the four strains, the size and kinetics of Ca2+ -dependent exocytosis, as well as the replenishment of synaptic vesicles, in IHCs was not affected. The degree of vesicle release at the fewer, but larger, individual remaining ribbon synapses colocalized with the post-synaptic afferent terminals is likely to increase, indicating the presence of a previously unknown degree of functional compensation in the ageing mouse cochlea.
Vestibular type I and type II hair cells and their afferent fibres send information to the brain regarding the position and movement of the head. The characteristic feature of type I hair cells is the expression of a low-voltage-activated outward rectifying K+ current, IK,L , whose biophysical properties and molecular identity are still largely unknown. In vitro, the afferent nerve calyx surrounding type I hair cells causes unstable intercellular K+ concentrations, altering the biophysical properties of IK,L . We found that in the absence of the calyx, IK,L in type I hair cells exhibited unique biophysical activation properties, which were faithfully reproduced by an allosteric channel gating scheme. These results form the basis for a molecular and pharmacological identification of IK,L .Type I and type II hair cells are the sensory receptors of the mammalian vestibular epithelia. Type I hair cells are characterized by their basolateral membrane being enveloped in a single large afferent nerve terminal, named the calyx, and by the expression of a low-voltage-activated outward rectifying K+ current, IK,L . The biophysical properties and molecular profile of IK,L are still largely unknown. By using the patch-clamp whole-cell technique, we examined the voltage- and time-dependent properties of IK,L in type I hair cells of the mouse semicircular canal. We found that the biophysical properties of IK,L were affected by an unstable K+ equilibrium potential (Veq K+ ). Both the outward and inward K+ currents shifted Veq K+ consistent with K+ accumulation or depletion, respectively, in the extracellular space, which we attributed to a residual calyx attached to the basolateral membrane of the hair cells. We therefore optimized the hair cell dissociation protocol in order to isolate mature type I hair cells without their calyx. In these cells, the uncontaminated IK,L showed a half-activation at -79.6 mV and a steep voltage dependence (2.8 mV). IK,L also showed complex activation and deactivation kinetics, which we faithfully reproduced by an allosteric channel gating scheme where the channel is able to open from all (five) closed states. The 'early' open states substantially contribute to IK,L activation at negative voltages. This study provides the first complete description of the 'native' biophysical properties of IK,L in adult mouse vestibular type I hair cells.
Hearing relies on the mechanosensory inner and outer hair cells (OHCs) of the organ of Corti, which convert mechanical deflections of their actin-rich stereociliary bundles into electrochemical signals.Several actin-associated proteins are essential for stereocilia formation and maintenance, and their absence leads to deafness.One of the most abundant actin-bundling proteins of stereocilia is plastin 1, but its function has never been directly assessed.Here, we found that plastin 1 knock-out (Pls1 KO) mice have a moderate and progressive form of hearing loss across all frequencies.Auditory hair cells developed normally in Pls1 KO, but in young adult animals, the stereocilia of inner hair cells were reduced in width and length.The stereocilia of OHCs were comparatively less affected; however, they also showed signs of degeneration in ageing mice.The hair bundle stiffness and the acquisition of the electrophysiological properties of hair cells were unaffected by the absence of plastin 1, except for a significant change in the adaptation properties, but not the size of the mechanoelectrical transducer currents.These results show that in contrast to other actin-bundling proteins such as espin, harmonin or Eps8, plastin 1 is dispensable for the initial formation of stereocilia.However, the progressive hearing loss and morphological defects of hair cells in adult Pls1 KO mice point at a specific role for plastin 1 in the preservation of adult stereocilia and optimal hearing.Hence, mutations in the human PLS1 gene may be associated with relatively mild and progressive forms of hearing loss.
'%3e%3cpath fill='%23012169' d='M0 0v30h60V0z'/%3e%3cpath stroke='%23fff' stroke-width='6' d='m0 0 60 30m0-30L0 30'/%3e%3cpath stroke='%23C8102E' stroke-width='4' d='m0 0 60 30m0-30L0 30' clip-path='url(%23b)'/%3e%3cpath stroke='%23fff' stroke-width='10' d='M30 0v30M0 15h60'/%3e%3cpath stroke='%23C8102E' stroke-width='6' d='M30 0v30M0 15h60'/%3e%3c/g%3e%3c/svg%3e)
