scholarly journals The effects of Tmc1 Beethoven mutation on mechanotransducer channel function in cochlear hair cells

2015 ◽  
Vol 146 (3) ◽  
pp. 233-243 ◽  
Author(s):  
Maryline Beurg ◽  
Adam C. Goldring ◽  
Robert Fettiplace
Keyword(s):  
Hair Cell ◽  
Hair Cells ◽  
Outer Hair Cells ◽  
Wild Type ◽  
Cochlear Hair Cells ◽  
Transmembrane Channel ◽  
Adaptive Shift ◽  
Electrical Transducer ◽  
Protein Isoform ◽  
The Relationship

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.

scholarly journals Developmental changes in the cochlear hair cell mechanotransducer channel and their regulation by transmembrane channel–like proteins

2012 ◽  
Vol 141 (1) ◽  
pp. 141-148 ◽  
Author(s):  
Kyunghee X. Kim ◽  
Robert Fettiplace
Keyword(s):  
Hair Cell ◽  
Hair Cells ◽  
Outer Hair Cells ◽  
Current Amplitude ◽  
Wild Type ◽  
Cochlear Hair Cells ◽  
Cochlear Hair Cell ◽  
Channel Current ◽  
Transmembrane Channel ◽  
Sound Detection

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 Ca2+ 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 Ca2+ permeability, PCa, of the OHC MT channel decreased from cochlear apex to base. This gradient in PCa was not apparent in IHCs and disappeared after P7 in OHCs. In Tmc1−/− mice, PCa in basal OHCs was larger than that in wild-type mice (to equal that of apical OHCs), whereas in Tmc2−/−, PCa in apical and basal OHCs and IHCs was decreased compared with that in wild-type mice. We postulate that differences in Ca2+ permeability reflect different subunit compositions of the MT channel determined by expression of Tmc1 and Tmc2, with the latter conferring higher PCa in IHCs and immature apical OHCs. Changes in PCa with maturation are consistent with a developmental decrease in abundance of Tmc2 in OHCs but not in IHCs.

scholarly journals Conductance and block of hair-cell mechanotransducer channels in transmembrane channel–like protein mutants

2014 ◽  
Vol 144 (1) ◽  
pp. 55-69 ◽  
Author(s):  
Maryline Beurg ◽  
Kyunghee X. Kim ◽  
Robert Fettiplace
Keyword(s):  
Hair Cells ◽  
Outer Hair Cells ◽  
Incomplete Block ◽  
Wild Type ◽  
Pharmacological Profile ◽  
Cochlear Hair Cells ◽  
Transmembrane Channel ◽  
Protein Mutants ◽  
Tip Link ◽  
Peptide Toxin

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.

scholarly journals Subunit determination of the conductance of hair-cell mechanotransducer channels

2014 ◽  
Vol 112 (5) ◽  
pp. 1589-1594 ◽  
Author(s):  
Maryline Beurg ◽  
Wei Xiong ◽  
Bo Zhao ◽  
Ulrich Müller ◽  
Robert Fettiplace
Keyword(s):  
Hair Cell ◽  
Outer Hair Cell ◽  
Fusion Partner ◽  
Wild Type ◽  
Cochlear Hair Cells ◽  
Transmembrane Channel ◽  
Unitary Conductance ◽  
Protein Isoform ◽  
Protocadherin 15

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.

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

2019 ◽  
Vol 116 (41) ◽  
pp. 20743-20749 ◽  
Author(s):  
Maryline Beurg ◽  
Amanda Barlow ◽  
David N. Furness ◽  
Robert Fettiplace
Keyword(s):  
Hair Cells ◽  
Single Channel ◽  
Maximum Amplitude ◽  
Outer Hair Cells ◽  
Single Amino Acid ◽  
Wild Type ◽  
Cochlear Hair Cells ◽  
Proximate Cause ◽  
Mechanoelectrical Transduction ◽  
Calcium Permeability

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.

scholarly journals Disruption of Hars2 in Cochlear Hair Cells Causes Progressive Mitochondrial Dysfunction and Hearing Loss in Mice

2021 ◽  
Vol 15 ◽  
Author(s):  
Pengcheng Xu ◽  
Longhao Wang ◽  
Hu Peng ◽  
Huihui Liu ◽  
Hongchao Liu ◽  
...  
Keyword(s):  
Hearing Loss ◽  
Hair Cell ◽  
Mitochondrial Dysfunction ◽  
Hair Cells ◽  
Cell Loss ◽  
Outer Hair Cells ◽  
Hair Cell Loss ◽  
Progressive Hearing Loss ◽  
Cochlear Hair Cells ◽  
Inner Hair Cells

Mutations in a number of genes encoding mitochondrial aminoacyl-tRNA synthetases lead to non-syndromic and/or syndromic sensorineural hearing loss in humans, while their cellular and physiological pathology in cochlea has rarely been investigated in vivo. In this study, we showed that histidyl-tRNA synthetase HARS2, whose deficiency is associated with Perrault syndrome 2 (PRLTS2), is robustly expressed in postnatal mouse cochlea including the outer and inner hair cells. Targeted knockout of Hars2 in mouse hair cells resulted in delayed onset (P30), rapidly progressive hearing loss similar to the PRLTS2 hearing phenotype. Significant hair cell loss was observed starting from P45 following elevated reactive oxygen species (ROS) level and activated mitochondrial apoptotic pathway. Despite of normal ribbon synapse formation, whole-cell patch clamp of the inner hair cells revealed reduced calcium influx and compromised sustained synaptic exocytosis prior to the hair cell loss at P30, consistent with the decreased supra-threshold wave I amplitudes of the auditory brainstem response. Starting from P14, increasing proportion of morphologically abnormal mitochondria was observed by transmission electron microscope, exhibiting swelling, deformation, loss of cristae and emergence of large intrinsic vacuoles that are associated with mitochondrial dysfunction. Though the mitochondrial abnormalities are more prominent in inner hair cells, it is the outer hair cells suffering more severe cell loss. Taken together, our results suggest that conditional knockout of Hars2 in mouse cochlear hair cells leads to accumulating mitochondrial dysfunction and ROS stress, triggers progressive hearing loss highlighted by hair cell synaptopathy and apoptosis, and is differentially perceived by inner and outer hair cells.

scholarly journals TMC1 is an essential component of a leak channel that modulates tonotopy and excitability of auditory hair cells in mice

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Shuang Liu ◽  
Shufeng Wang ◽  
Linzhi Zou ◽  
Jie Li ◽  
Chenmeng Song ◽  
...  
Keyword(s):  
Hair Cells ◽  
Dependent Manner ◽  
Biological Processes ◽  
Action Potential Firing ◽  
Cochlear Hair Cells ◽  
Auditory Hair Cells ◽  
Leak Channel ◽  
Transmembrane Channel ◽  
Electrical Transducer ◽  
Potential Firing

Hearing sensation relies on the mechano-electrical transducer (MET) channel of cochlear hair cells, in which transmembrane channel-like 1 (TMC1) and transmembrane channel-like 2 (TMC2) have been proposed to be the pore-forming subunits in mammals. TMCs were also found to regulate biological processes other than MET in invertebrates, ranging from sensations to motor function. However, whether TMCs have a non-MET role remains elusive in mammals. Here, we report that in mouse hair cells, TMC1, but not TMC2, provides a background leak conductance, with properties distinct from those of the MET channels. By cysteine substitutions in TMC1, we characterized four amino acids that are required for the leak conductance. The leak conductance is graded in a frequency-dependent manner along the length of the cochlea and is indispensable for action potential firing. Taken together, our results show that TMC1 confers a background leak conductance in cochlear hair cells, which may be critical for the acquisition of sound-frequency and -intensity.

scholarly journals TMC1 Confers a Leak Conductance to Modulate Excitability of Auditory Hair Cells in Mammals

2019 ◽  
Author(s):  
Shuang Liu ◽  
Shufeng Wang ◽  
Linzhi Zou ◽  
Jie Li ◽  
Chenmeng Song ◽  
...  
Keyword(s):  
Hair Cells ◽  
Biological Processes ◽  
Membrane Excitability ◽  
Action Potential Firing ◽  
Cochlear Hair Cells ◽  
Auditory Hair Cells ◽  
Transmembrane Channel ◽  
Electrical Transducer ◽  
Potential Firing ◽  
Open Question

ABSTRACTHearing sensation relies on the mechano-electrical transducer (MET) channel of cochlear hair cells, in which Transmembrane channel-like 1 (TMC1) and TMC2 have been proposed to be the pore-forming subunits. Meanwhile it has been reported that TMCs regulate other biological processes in a variety of lower organisms ranging from sensations to motor functions. However, it is still an open question whether TMCs play roles other than their function in MET in mammals. In this study, we report that in mouse hair cells TMC1, but not TMC2, provides a background leak conductance, with properties distinct from those of the MET channels. By cysteine substitution, 4 amino acids of TMC1 are characterized critical for the leak conductance. The leak conductance is essential for action potential firing and tonotopic along the cochlear coil. Taken together, our results suggest that TMC1 confers a background leak conductance that modulates membrane excitability in cochlear hair cells.

scholarly journals Attempted rescue of circling mice by hair cell-specific expression (Myo7a promoter) of tmie transgene

2017 ◽  
Vol 73 (7) ◽  
pp. 399-403
Author(s):  
Seojin Park ◽  
Soyoung Jang ◽  
Jeong-Han Lee ◽  
Sung-Hyun Kim ◽  
Zae Young Ryoo
Keyword(s):  
Hair Cell ◽  
Hair Cells ◽  
Fluorescent Protein ◽  
Outer Hair Cells ◽  
Abnormal Behavior ◽  
Spontaneous Mutant ◽  
Wild Type ◽  
Specific Expression ◽  
Brainstem Response ◽  
Ganglion Neurons

The spontaneous mutant circling mouse (cir/cir) is deaf and displays abnormal behavior, particularly circling and head tossing. To rescue the circling mouse phenotype, we produced transgenic mice with hair cell-specific expression of the transmembrane inner ear (tmie) gene, using the Myo7a promoter, and generated cir/cir homozygous mice carrying the transgene (cir/cir-tg<sup>Myo7a</sup>) by breeding with circling mice. The cir/cir-tg<sup>Myo7a</sup> mice still exhibited circling behavior and were unable to swim in water unlike cir/cir-tg<sup>CMV</sup> mice and wild-type mice. An auditory brainstem response (ABR) test demonstrated that the cir/cir-tgMyo7a mice could not respond to sound. Immunohistochemical analysis showed enhanced green fluorescent protein, a marker of tmie expression, in the inner and outer hair cells of the cir/cir-tg<sup>Myo7a</sup> mice. Hair cells and spiral ganglion neurons in the cir/cir-tg<sup>Myo7a</sup> mice were recovered, but not completely. This study demonstrates that tmie transgene expression in hair cells alone could not restore wild-type hearing and behavior in circling mice.

scholarly journals Characterization of HA-tagged α9 and α10 nAChRs in the mouse cochlea

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Pankhuri Vyas ◽  
Megan Beers Wood ◽  
Yuanyuan Zhang ◽  
Adam C. Goldring ◽  
Fatima-Zahra Chakir ◽  
...  
Keyword(s):  
Nicotinic Acetylcholine Receptors ◽  
Hair Cells ◽  
Outer Hair Cells ◽  
Synaptic Function ◽  
Auditory Brainstem Responses ◽  
Wild Type ◽  
Cochlear Hair Cells ◽  
Heterozygous Condition ◽  
Adult Mice ◽  
Efferent Neurons

AbstractNeurons of the medial olivary complex inhibit cochlear hair cells through the activation of α9α10-containing nicotinic acetylcholine receptors (nAChRs). Efforts to study the localization of these proteins have been hampered by the absence of reliable antibodies. To overcome this obstacle, CRISPR-Cas9 gene editing was used to generate mice in which a hemagglutinin tag (HA) was attached to the C-terminus of either α9 or α10 proteins. Immunodetection of the HA tag on either subunit in the organ of Corti of adult mice revealed immunopuncta clustered at the synaptic pole of outer hair cells. These puncta were juxtaposed to immunolabeled presynaptic efferent terminals. HA immunopuncta also occurred in inner hair cells of pre-hearing (P7) but not in adult mice. These immunolabeling patterns were similar for both homozygous and heterozygous mice. All HA-tagged genotypes had auditory brainstem responses not significantly different from those of wild type littermates. The activation of efferent neurons in heterozygous mice evoked biphasic postsynaptic currents not significantly different from those of wild type hair cells. However, efferent synaptic responses were significantly smaller and less frequent in the homozygous mice. We show that HA-tagged nAChRs introduced in the mouse by a CRISPR knock-in are regulated and expressed like the native protein, and in the heterozygous condition mediate normal synaptic function. The animals thus generated have clear advantages for localization studies.

Cochlear Hair Cell Stereocilia Loss in LP/J Mice with Bone Dysplasia of the Middle Ear

1989 ◽  
Vol 98 (6) ◽  
pp. 461-465 ◽  
Author(s):  
Richard A. Chole ◽  
Maggie Chiu
Keyword(s):  
Hair Cell ◽  
Middle Ear ◽  
Hair Cells ◽  
Inbred Mice ◽  
Bone Dysplasia ◽  
Cell Loss ◽  
Outer Hair Cells ◽  
Cochlear Hair Cells ◽  
Inner Hair Cells ◽  
Cochlear Hair Cell

LP/J inbred mice spontaneously develop bony lesions of the middle ear and otic capsule that are similar to those of human otosclerosis and tympanosclerosis. These mice also have progressive loss of hearing due to cochlear hair cell loss. The purpose of this study was to describe quantitatively the deterioration and loss of cochlear hair cells to serve as a basis for future experiments attempting to alter the course of this disorder. Cochleas from 37 LP/J inbred mice were examined by scanning electron microscopy. The stereocilia loss in the cochlea was evident as early as 15 weeks of age and progressed from the basal turn to the apex. Outer hair cells were affected more than inner hair cells. As outer hair cells deteriorated we observed fusion, bending, and breakage of stereocilia. There were no apparent differences in the mode of deterioration among the three rows of outer hair cells. Stereocilia fusion of inner hair cells occurred at an older age, and giant, elongated stereocilia were found in some of the animals.

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