scholarly journals Deafness mutation D572N of TMC1 destabilizes TMC1 expression by disrupting LHFPL5 binding

2020 ◽  
Vol 117 (47) ◽  
pp. 29894-29903
Author(s):  
Xiaojie Yu ◽  
Qirui Zhao ◽  
Xiaofen Li ◽  
Yixuan Chen ◽  
Ye Tian ◽  
...  
Keyword(s):  
Single Molecule ◽  
Hair Cells ◽  
Macromolecular Complex ◽  
Fusion Partner ◽  
Functional Interactions ◽  
Transmembrane Channel ◽  
Physical Organization ◽  
Heterologous Expression Systems ◽  
Critical Components ◽  
Rare Cells

Transmembrane channel-like protein 1 (TMC1) and lipoma HMGIC fusion partner-like 5 (LHFPL5) are recognized as two critical components of the mechanotransduction complex in inner-ear hair cells. However, the physical and functional interactions of TMC1 and LHFPL5 remain largely unexplored. We examined the interaction between TMC1 and LHFPL5 by using multiple approaches, including our recently developed ultrasensitive microbead-based single-molecule pulldown (SiMPull) assay. We demonstrate that LHFPL5 physically interacts with and stabilizes TMC1 in both heterologous expression systems and in the soma and hair bundle of hair cells. Moreover, the semidominant deafness mutation D572N in human TMC1 (D569N in mouse TMC1) severely disrupted LHFPL5 binding and destabilized TMC1 expression. Thus, our findings reveal previously unrecognized physical and functional interactions of TMC1 and LHFPL5 and provide insights into the molecular mechanism by which the D572N mutation causes deafness. Notably, these findings identify a missing link in the currently known physical organization of the mechanotransduction macromolecular complex. Furthermore, this study has demonstrated the power of the microbead-based SiMPull assay for biochemical investigation of rare cells such as hair cells.

scholarly journals Treadmilling FtsZ polymers drive the directional movement of sPG-synthesis enzymes via a Brownian ratchet mechanism

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Joshua W. McCausland ◽  
Xinxing Yang ◽  
Georgia R. Squyres ◽  
Zhixin Lyu ◽  
Kevin E. Bruce ◽  
...  
Keyword(s):  
Cell Wall ◽  
Single Molecule ◽  
Theoretical Modelling ◽  
Binding Potential ◽  
Central Component ◽  
Macromolecular Complex ◽  
Bacterial Cells ◽  
Brownian Ratchet ◽  
Ratchet Mechanism ◽  
Directional Movement

AbstractThe FtsZ protein is a central component of the bacterial cell division machinery. It polymerizes at mid-cell and recruits more than 30 proteins to assemble into a macromolecular complex to direct cell wall constriction. FtsZ polymers exhibit treadmilling dynamics, driving the processive movement of enzymes that synthesize septal peptidoglycan (sPG). Here, we combine theoretical modelling with single-molecule imaging of live bacterial cells to show that FtsZ’s treadmilling drives the directional movement of sPG enzymes via a Brownian ratchet mechanism. The processivity of the directional movement depends on the binding potential between FtsZ and the sPG enzyme, and on a balance between the enzyme’s diffusion and FtsZ’s treadmilling speed. We propose that this interplay may provide a mechanism to control the spatiotemporal distribution of active sPG enzymes, explaining the distinct roles of FtsZ treadmilling in modulating cell wall constriction rate observed in different bacteria.

scholarly journals LRRC52 regulates BK channel function and localization in mouse cochlear inner hair cells

2019 ◽  
Vol 116 (37) ◽  
pp. 18397-18403 ◽  
Author(s):  
Christopher J. Lingle ◽  
Pedro L. Martinez-Espinosa ◽  
Aizhen Yang-Hood ◽  
Luis E. Boero ◽  
Shelby Payne ◽  
...  
Keyword(s):  
Hair Cells ◽  
Glutamate Release ◽  
Nerve Fibers ◽  
Bk Channels ◽  
Bk Channel ◽  
Membrane Potentials ◽  
Macromolecular Complex ◽  
Inner Hair Cells ◽  
Channel Function ◽  
Voltage Dependent

The perception of sound relies on sensory hair cells in the cochlea that convert the mechanical energy of sound into release of glutamate onto postsynaptic auditory nerve fibers. The hair cell receptor potential regulates the strength of synaptic transmission and is shaped by a variety of voltage-dependent conductances. Among these conductances, the Ca2+- and voltage-activated large conductance Ca2+-activated K+channel (BK) current is prominent, and in mammalian inner hair cells (IHCs) displays unusual properties. First, BK currents activate at unprecedentedly negative membrane potentials (−60 mV) even in the absence of intracellular Ca2+elevations. Second, BK channels are positioned in clusters away from the voltage-dependent Ca2+channels that mediate glutamate release from IHCs. Here, we test the contributions of two recently identified leucine-rich-repeat–containing (LRRC) regulatory γ subunits, LRRC26 and LRRC52, to BK channel function and localization in mouse IHCs. Whereas BK currents and channel localization were unaltered in IHCs fromLrrc26knockout (KO) mice, BK current activation was shifted more than +200 mV in IHCs fromLrrc52KO mice. Furthermore, the absence of LRRC52 disrupted BK channel localization in the IHCs. Given that heterologous coexpression of LRRC52 with BK α subunits shifts BK current gating about −90 mV, to account for the profound change in BK activation range caused by removal of LRRC52, we suggest that additional factors may help define the IHC BK gating range. LRRC52, through stabilization of a macromolecular complex, may help retain some other components essential both for activation of BK currents at negative membrane potentials and for appropriate BK channel positioning.

scholarly journals Mechanical gating of the auditory transduction channel TMC1 involves the fourth and sixth transmembrane helices

2022 ◽  
Author(s):  
Nurunisa Akyuz ◽  
K. Domenica Karavitaki ◽  
Bifeng Pan ◽  
Panos I. Tamvakologos ◽  
Kelly P. Brock ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Hair Cells ◽  
Single Channel ◽  
Transmembrane Helices ◽  
Open Probability ◽  
Transmembrane Channel ◽  
Force Sensitivity ◽  
Electrophysiological Recordings ◽  
Auditory Transduction ◽  
Pore Lining

The transmembrane channel-like (TMC) 1 and 2 proteins play a central role in auditory transduction, forming ion channels that convert sound into electrical signals. However, the molecular mechanism of their gating remains unknown. Here, using predicted structural models as a guide, we probed the effects of twelve mutations on the mechanical gating of the transduction currents in native hair cells of Tmc1/2-null mice expressing virally introduced TMC1 variants. Whole-cell electrophysiological recordings revealed that mutations within the pore-lining transmembrane (TM) helices 4 and 6 modified gating, reducing the force sensitivity or shifting the open probability of the channels, or both. For some of the mutants, these changes were accompanied by a change in single-channel conductance. Our observations are in line with a model wherein conformational changes in the TM4 and TM6 helices are involved in the mechanical gating of the transduction channel.

scholarly journals Chromatography-Free Purification Strategies for Large Biological Macromolecular Complexes Involving Fractionated PEG Precipitation and Density Gradients

Life ◽  
2021 ◽  
Vol 11 (12) ◽  
pp. 1289
Author(s):  
Fabian Henneberg ◽  
Ashwin Chari
Keyword(s):  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Macromolecular Complex ◽  
Biological Macromolecules ◽  
Chromatographic Purification ◽  
Complex Interplay ◽  
Macromolecular Complexes ◽  
Functional Interactions ◽  
Biotechnological Processes ◽  
Essential Prerequisite

A complex interplay between several biological macromolecules maintains cellular homeostasis. Generally, the demanding chemical reactions which sustain life are not performed by individual macromolecules, but rather by several proteins that together form a macromolecular complex. Understanding the functional interactions amongst subunits of these macromolecular machines is fundamental to elucidate mechanisms by which they maintain homeostasis. As the faithful function of macromolecular complexes is essential for cell survival, their mis-function leads to the development of human diseases. Furthermore, detailed mechanistic interrogation of the function of macromolecular machines can be exploited to develop and optimize biotechnological processes. The purification of intact macromolecular complexes is an essential prerequisite for this; however, chromatographic purification schemes can induce the dissociation of subunits or the disintegration of the whole complex. Here, we discuss the development and application of chromatography-free purification strategies based on fractionated PEG precipitation and orthogonal density gradient centrifugation that overcomes existing limitations of established chromatographic purification protocols. The presented case studies illustrate the capabilities of these procedures for the purification of macromolecular complexes.

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 The Mechanosensory Transduction Machinery in Inner Ear Hair Cells

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.

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 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.

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