Tip-link protein protocadherin 15 interacts with transmembrane channel-like proteins TMC1 and TMC2

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 Mechanically Weak Extracellular Membrane-Adjacent Domain Induces Dimerization of Protocadherin-15

10.1101/460097 ◽

2018 ◽

Author(s):

P. De-la-Torre ◽

D. Choudhary ◽

R. Araya-Secchi ◽

Y. Narui ◽

M. Sotomayor

Keyword(s):

Protein Interactions ◽

Analytical Ultracentrifugation ◽

Size Exclusion ◽

Molecular Architecture ◽

Protein Protein Interactions ◽

Tip Link ◽

Exclusion Chromatography ◽

Protocadherin 15 ◽

Cadherin 23 ◽

Dynamics Simulations

ABSTRACTThe cadherin superfamily of proteins is defined by the presence of extracellular cadherin (EC) repeats that engage in protein-protein interactions to mediate cell-cell adhesion, cell signaling, and mechanotransduction. The extracellular domains of non-classical cadherins often have a large number of EC repeats along with other subdomains of various folds. Protocadherin-15 (PCDH15), a protein component of the inner-ear tip link filament essential for mechanotransduction, has eleven EC repeats and a membrane adjacent domain (MAD12) of atypical fold. Here we report the crystal structure of a pig PCDH15 fragment including EC10, EC11, and MAD12 in a parallel dimeric arrangement. MAD12 has a unique molecular architecture and folds as a ferredoxin-like domain similar to that found in the nucleoporin protein Nup54. Analytical ultracentrifugation experiments along with size exclusion chromatography coupled to multi-angle laser light scattering and small-angle X-ray scattering corroborate the crystallographic dimer and show that MAD12 induces parallel dimerization of PCDH15 near its membrane insertion point. In addition, steered molecular dynamics simulations suggest that MAD12 is mechanically weak and may unfold before tip-link rupture. Sequence analyses and structural modeling predict the existence of similar domains in cadherin-23, protocadherin-24, and the “giant” FAT and CELSR cadherins, indicating that some of them may also exhibit MAD-induced parallel dimerization.

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The tip link protein Cadherin-23: From Hearing Loss to Cancer

Pharmacological Research ◽

10.1016/j.phrs.2018.01.026 ◽

2018 ◽

Vol 130 ◽

pp. 25-35 ◽

Cited By ~ 4

Author(s):

Paridhy Vanniya. S ◽

C.R. Srikumari Srisailapathy ◽

Ramkumar Kunka Mohanram

Keyword(s):

Hearing Loss ◽

Link Protein ◽

Tip Link ◽

Cadherin 23

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Weakening of Interaction Networks with Aging in Tip-Link Protein Induces Hearing Loss

Biochemical Journal ◽

10.1042/bcj20200799 ◽

2020 ◽

Author(s):

Surbhi Garg ◽

Amin Sagar ◽

Gayathri S. Singaraju ◽

Rahul Dani ◽

Naimat Kalim Bari ◽

...

Keyword(s):

Hearing Loss ◽

Molecular Feature ◽

Link Protein ◽

Correlated Motions ◽

Age Related ◽

Disease Mutations ◽

Tip Link ◽

Bond Network ◽

Cadherin 23 ◽

Age Related Hearing Loss

Age-related hearing loss (ARHL) is a common condition in humans marking the gradual decrease in hearing with age. Perturbations in the tip-link protein cadherin-23 that absorbs the mechanical tension from sound and maintains the integrity of hearing is associated with ARHL. Here, in search of molecular origins for ARHL, we dissect the conformational behavior of cadherin-23 along with the mutant S47P that progresses the hearing-loss drastically. Using an array of experimental and computational approaches, we highlight a lower thermodynamic stability, significant weakening in the hydrogen-bond network and inter-residue correlations among β-strands, due to the S47P mutation. The loss in correlated motions translates to not only a remarkable two orders of magnitude slower folding in the mutant but also to a proportionately complex unfolding mechanism. We thus propose that loss in correlated motions within cadherin-23 with aging may trigger ARHL, a molecular feature that likely holds true for other disease-mutations in β-strand-rich proteins.

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Faculty Opinions recommendation of Cadherin 23 and protocadherin 15 interact to form tip-link filaments in sensory hair cells.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1092088.544638 ◽

2007 ◽

Author(s):

Peter Gillespie

Keyword(s):

Hair Cells ◽

Sensory Hair ◽

Sensory Hair Cells ◽

Tip Link ◽

Protocadherin 15 ◽

Cadherin 23

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Subunit determination of the conductance of hair-cell mechanotransducer channels

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1420906112 ◽

2014 ◽

Vol 112 (5) ◽

pp. 1589-1594 ◽

Cited By ~ 77

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.

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Faculty Opinions recommendation of Structure of mouse protocadherin 15 of the stereocilia tip link in complex with LHFPL5.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.733748691.793580180 ◽

2020 ◽

Author(s):

Tobias Moser

Keyword(s):

Tip Link ◽

Protocadherin 15

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Structural relationship between the putative hair cell mechanotransduction channel TMC1 and TMEM16 proteins

eLife ◽

10.7554/elife.38433 ◽

2018 ◽

Vol 7 ◽

Cited By ~ 35

Author(s):

Angela Ballesteros ◽

Cristina Fenollar-Ferrer ◽

Kenton Jon Swartz

Keyword(s):

Hair Cell ◽

Hair Cells ◽

Genetic Alterations ◽

Structural Relationship ◽

Large Cavity ◽

X Ray ◽

Molecular Identity ◽

Transmembrane Channel ◽

Tip Link ◽

Channel Properties

The hair cell mechanotransduction (MET) channel complex is essential for hearing, yet it’s molecular identity and structure remain elusive. The transmembrane channel–like 1 (TMC1) protein localizes to the site of the MET channel, interacts with the tip-link responsible for mechanical gating, and genetic alterations in TMC1 alter MET channel properties and cause deafness, supporting the hypothesis that TMC1 forms the MET channel. We generated a model of TMC1 based on X-ray and cryo-EM structures of TMEM16 proteins, revealing the presence of a large cavity near the protein-lipid interface that also harbors the Beethoven mutation, suggesting that it could function as a permeation pathway. We also find that hair cells are permeable to 3 kDa dextrans, and that dextran permeation requires TMC1/2 proteins and functional MET channels, supporting the presence of a large permeation pathway and the hypothesis that TMC1 is a pore forming subunit of the MET channel complex.

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