Subunit determination of the conductance of hair-cell mechanotransducer channels

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.

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

The Journal of General Physiology ◽

10.1085/jgp.201511458 ◽

2015 ◽

Vol 146 (3) ◽

pp. 233-243 ◽

Cited By ~ 24

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.

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

The Journal of General Physiology ◽

10.1085/jgp.201210913 ◽

2012 ◽

Vol 141 (1) ◽

pp. 141-148 ◽

Cited By ~ 59

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.

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The Mechanosensory Transduction Machinery in Inner Ear Hair Cells

Annual Review of Biophysics ◽

10.1146/annurev-biophys-062420-081842 ◽

2020 ◽

Vol 50 (1) ◽

Author(s):

Wang Zheng ◽

Jeffrey R. Holt

Keyword(s):

Hair Cell ◽

Inner Ear ◽

Hair Cells ◽

Annual Review ◽

Publication Date ◽

Fusion Partner ◽

Mechanical Stimuli ◽

Physical Stimulation ◽

Mechanosensory Transduction ◽

Protocadherin 15

Sound-induced mechanical stimuli are detected by elaborate mechanosensory transduction (MT) machinery in highly specialized hair cells of the inner ear. Genetic studies of inherited deafness in the past decades have uncovered several molecular constituents of the MT complex, and intense debate has surrounded the molecular identity of the pore-forming subunits. How the MT components function in concert in response to physical stimulation is not fully understood. In this review, we summarize and discuss multiple lines of evidence supporting the hypothesis that transmembrane channel-like 1 is a long-sought MT channel subunit. We also review specific roles of other components of the MT complex, including protocadherin 15, cadherin 23, lipoma HMGIC fusion partner-like 5, transmembrane inner ear, calcium and integrin-binding family member 2, and ankyrins. Based on these recent advances, we propose a unifying theory of hair cell MT that may reconcile most of the functional discoveries obtained to date. Finally, we discuss key questions that need to be addressed for a comprehensive understanding of hair cell MT at molecular and atomic levels. Expected final online publication date for the Annual Review of Biophysics, Volume 50 is May 6, 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

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

The Journal of General Physiology ◽

10.1085/jgp.201411173 ◽

2014 ◽

Vol 144 (1) ◽

pp. 55-69 ◽

Cited By ~ 49

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.

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A gap-junction mutation in the mouse cochlea reveals cochlear amplification is driven by outer hair cell extracellular receptor potentials

10.1101/2021.08.11.455955 ◽

2021 ◽

Author(s):

Snezana Levic ◽

Victoria A Lukashkina ◽

Patricio Simoes ◽

Andrei N Lukashkin ◽

Ian J Russell

Keyword(s):

Hair Cell ◽

Gap Junction ◽

High Frequency ◽

Outer Hair Cell ◽

Stria Vascularis ◽

Organ Of Corti ◽

Receptor Potential ◽

Wild Type ◽

Hearing Sensitivity ◽

Cochlear Amplification

Cochlear amplification, whereby cochlear responses to low-to-moderate sound levels are 31 amplified and compressed to loud sounds, is attributed to outer hair cell (OHC) electromotility 32 driven by voltage changes across the OHC basolateral membranes due to sound-induced 33 receptor-current modulation. Cochlear operation at high acoustic frequencies is enigmatic 34 because the OHC intracellular receptor potential (RP) is severely attenuated at these 35 frequencies. Clues to understanding the voltage control of OHC electromotility at different 36 frequencies was provided by measurements from CD-1 mice with an A88V mutation of the 37 gap-junction (GJ) protein connexin 30 (Cx30), which with Cx26, form heterogeneous GJs 38 between supporting cells in the organ of Corti (OoC) and stria vascularis. The A88V mutation 39 results in a smaller GJ conductance which may explain why the resistance across the OoC in 40 CD-1Cx30A88V/A88V mutants is higher compared with wild-type mice. The endocochlear 41 potential, which drives the OHC receptor current and, consequently, the OHC RPs, is smaller 42 in CD-1Cx30A88V/A88V mutants. Even so, their high-frequency hearing sensitivity equals that of 43 wild-type mice. Preservation of high-frequency hearing correlates with similar amplitude of 44 extracellular receptor potentials (ERPs), measured immediately adjacent to the OHCs. ERPs 45 are generated through OHC receptor current flow across the OoC electrical resistance, which 46 is larger in CD-1Cx30A88V/A88V than in wild-type mice. Thus, smaller OHC receptor currents 47 flowing across a larger OoC resistance in CD-1Cx30A88V/A88V mice may explain why their ERP 48 magnitudes are similar to wild-type mice. It is proposed that the ERPs, which are not subject 49 to low-pass electrical filtering, drive high-frequency cochlear amplification.

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Codeficiency of Lysosomal Mucolipins 3 and 1 in Cochlear Hair Cells Diminishes Outer Hair Cell Longevity and Accelerates Age-Related Hearing Loss

Journal of Neuroscience ◽

10.1523/jneurosci.3368-17.2018 ◽

2018 ◽

Vol 38 (13) ◽

pp. 3177-3189 ◽

Cited By ~ 2

Author(s):

Teerawat Wiwatpanit ◽

Natalie N. Remis ◽

Aisha Ahmad ◽

Yingjie Zhou ◽

John C. Clancy ◽

...

Keyword(s):

Hearing Loss ◽

Hair Cell ◽

Hair Cells ◽

Outer Hair Cell ◽

Cochlear Hair Cells ◽

Age Related ◽

Age Related Hearing Loss ◽

Cell Longevity

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The role of transmembrane channel–like proteins in the operation of hair cell mechanotransducer channels

The Journal of General Physiology ◽

10.1085/jgp.201311068 ◽

2013 ◽

Vol 142 (5) ◽

pp. 493-505 ◽

Cited By ~ 63

Author(s):

Kyunghee X. Kim ◽

Maryline Beurg ◽

Carole M. Hackney ◽

David N. Furness ◽

Shanthini Mahendrasingam ◽

...

Keyword(s):

Hair Cells ◽

Hair Bundle ◽

Wild Type ◽

Cochlear Hair Cells ◽

Transmembrane Channel ◽

Protein Partners ◽

Tip Link ◽

Lower Permeability ◽

Precise Role

Sound stimuli elicit movement of the stereocilia that make up the hair bundle of cochlear hair cells, putting tension on the tip links connecting the stereocilia and thereby opening mechanotransducer (MT) channels. Tmc1 and Tmc2, two members of the transmembrane channel–like family, are necessary for mechanotransduction. To assess their precise role, we recorded MT currents elicited by hair bundle deflections in mice with null mutations of Tmc1, Tmc2, or both. During the first postnatal week, we observed a normal MT current in hair cells lacking Tmc1 or Tmc2; however, in the absence of both isoforms, we recorded a large MT current that was phase-shifted 180°, being evoked by displacements of the hair bundle away from its tallest edge rather than toward it as in wild-type hair cells. The anomalous MT current in hair cells lacking Tmc1 and Tmc2 was blocked by FM1-43, dihydrostreptomycin, and extracellular Ca2+ at concentrations similar to those that blocked wild type. MT channels in the double knockouts carried Ca2+ with a lower permeability than wild-type or single mutants. The MT current in double knockouts persisted during exposure to submicromolar Ca2+, even though this treatment destroyed the tip links. We conclude that the Tmc isoforms do not themselves constitute the MT channel but are essential for targeting and interaction with the tip link. Changes in the MT conductance and Ca2+ permeability observed in the absence of Tmc1 mutants may stem from loss of interaction with protein partners in the transduction complex.

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Knock-In Mice with Myo3a Y137C Mutation Displayed Progressive Hearing Loss and Hair Cell Degeneration in the Inner Ear

Neural Plasticity ◽

10.1155/2018/4372913 ◽

2018 ◽

Vol 2018 ◽

pp. 1-10 ◽

Cited By ~ 1

Author(s):

Peipei Li ◽

Zongzhuang Wen ◽

Guangkai Zhang ◽

Aizhen Zhang ◽

Xiaolong Fu ◽

...

Keyword(s):

Hair Cell ◽

Inner Ear ◽

Hair Cells ◽

Critical Role ◽

Kinase Domain ◽

Mutant Mice ◽

Structural Abnormality ◽

Wild Type ◽

Cell Degeneration ◽

Cochlear Hair Cells

Myo3a is expressed in cochlear hair cells and retinal cells and is responsible for human recessive hereditary nonsyndromic deafness (DFNB30). To investigate the mechanism of DFNB30-type deafness, we established a mouse model of Myo3a kinase domain Y137C mutation by using CRISPR/Cas9 system. No difference in hearing between 2-month-old Myo3a mutant mice and wild-type mice was observed. The hearing threshold of the ≥6-month-old mutant mice was significantly elevated compared with that of the wild-type mice. We observed degeneration in the inner ear hair cells of 6-month-old Myo3a mutant mice, and the degeneration became more severe at the age of 12 months. We also found structural abnormality in the cochlear hair cell stereocilia. Our results showed that Myo3a is essential for normal hearing by maintaining the intact structure of hair cell stereocilia, and the kinase domain plays a critical role in the normal functions of Myo3a. This mouse line is an excellent model for studying DFNB30-type deafness in humans.

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