Cysteine Substitution Reveals the Pore-Forming Region of TMC1 in Hair Cell Sensory Transduction Channels

Most genetic diseases arise from recessive point mutations that require correction, rather than disruption, of the pathogenic allele to benefit patients. Base editing has the potential to directly repair point mutations and provide therapeutic restoration of gene function. Mutations of transmembrane channel-like 1 gene (TMC1) can cause dominant or recessive deafness. We developed a base editing strategy to treat Baringo mice, which carry a recessive, loss-of-function point mutation (c.A545G; resulting in the substitution p.Y182C) in Tmc1 that causes deafness. Tmc1 encodes a protein that forms mechanosensitive ion channels in sensory hair cells of the inner ear and is required for normal auditory function. We found that sensory hair cells of Baringo mice have a complete loss of auditory sensory transduction. To repair the mutation, we tested several optimized cytosine base editors (CBEmax variants) and guide RNAs in Baringo mouse embryonic fibroblasts. We packaged the most promising CBE, derived from an activation-induced cytidine deaminase (AID), into dual adeno-associated viruses (AAVs) using a split-intein delivery system. The dual AID-CBEmax AAVs were injected into the inner ears of Baringo mice at postnatal day 1. Injected mice showed up to 51% reversion of the Tmc1 c.A545G point mutation to wild-type sequence (c.A545A) in Tmc1 transcripts. Repair of Tmc1 in vivo restored inner hair cell sensory transduction and hair cell morphology and transiently rescued low-frequency hearing 4 weeks after injection. These findings provide a foundation for a potential one-time treatment for recessive hearing loss and support further development of base editing to correct pathogenic point mutations.

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Channels and electrical signals

Sensory Transduction ◽

10.1093/oso/9780198835028.003.0003 ◽

2019 ◽

pp. 37-56

Author(s):

Gordon L. Fain

Keyword(s):

Ion Channels ◽

Hair Cell ◽

Ion Selectivity ◽

Ion Homeostasis ◽

Membrane Conductance ◽

Sensory Transduction ◽

Ionotropic Receptor ◽

Electrical Signals ◽

Voltage Clamping ◽

And Function

“Channels and electrical signals” is the third chapter of the book Sensory Transduction and reviews the structure and function of ion channels, the structure of channel pores, and mechanisms of gating. It introduces ionotropic receptor molecules, which are proteins that function as sensory receptors but are also ion channels, whose gating can produce changes in membrane conductance directly. It then uses the hair cell of the inner ear as an example to introduce the concepts of membrane potentials, the Nernst equation, ion homeostasis, the Goldman voltage equation, and driving force. A description of the technique of voltage clamping follows, together with the application of this technique to the hair cell to explain the method of measuring changes in channel gating and the ion selectivity of channel pores.

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Adaptive cell invasion maintains organ homeostasis

10.1101/2020.12.01.404954 ◽

2020 ◽

Author(s):

Julia Peloggia ◽

Daniela Münch ◽

Paloma Meneses-Giles ◽

Andrés Romero-Carvajal ◽

Melainia McClain ◽

...

Keyword(s):

Hair Cell ◽

Lateral Line ◽

Cell Invasion ◽

Hair Cells ◽

Environmental Changes ◽

Cell Function ◽

Ionic Composition ◽

Sensory Transduction ◽

Aqueous Environment

Mammalian inner ear and fish lateral line sensory hair cells depend on fluid motion to transduce environmental signals and elicit a response. In mammals, actively maintained ionic homeostasis of the cochlear and vestibular fluid (endolymph) is essential for hair cell function and numerous mammalian hearing and vestibular disorders arise from disrupted endolymph ion homeostasis. Lateral line hair cells, however, are openly exposed to the aqueous environment with fluctuating ionic composition. How sensory transduction in the lateral line is maintained during environmental changes of ionic composition is not fully understood. Using lineage labeling, in vivo time lapse imaging and scRNA-seq, we discovered highly motile skin-derived cells that invade mature mechanosensory organs of the zebrafish lateral line and differentiate into Neuromast-associated (Nm) ionocytes. Furthermore, the invasive behavior is adaptive as it is triggered by drastic fluctuations in environmental stimuli. Our findings challenge the notion of an entirely placodally-derived lateral line and identify Nm ionocytes as regulators of mechanosensory hair cell function by modulating the ionic microenvironment. The discovery of lateral line ionocytes provides an experimentally accessible in vivo system to study cell invasion and migration, as well as the physiological adaptation of vertebrate organs to changing environmental conditions.

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TRPV5, TRPV6, TRPM6, and TRPM7 do not contribute to hair-cell mechanotransduction

10.1101/204172 ◽

2017 ◽

Cited By ~ 1

Author(s):

Clive P. Morgan ◽

Hongyu Zhao ◽

Meredith LeMasurier ◽

Wei Xiong ◽

Bifeng Pan ◽

...

Keyword(s):

Hair Cell ◽

Hair Cells ◽

Auditory Function ◽

Cochlear Hair Cells ◽

Cochlear Hair Cell ◽

Cysteine Substitution ◽

Tip Link ◽

Transient Receptor ◽

Receptor Channel

AbstractThe hair-cell mechanotransduction channel remains unidentified. We tested whether four transient receptor channel (TRP) family members, TRPV5, TRPV6, TRPM6, and TRPM7, participated in transduction. Using cysteine-substitution mouse knock-ins and methanethiosulfonate reagents selective for those alleles, we found that inhibition of TRPV5 or TRPV6 had no effect on transduction in mouse cochlear hair cells. TRPM6 and TRPM7 each interacted with the tip-link component PCDH15 in cultured eukaryotic cells, which suggested they could participate in transduction. Cochlear hair cell transduction was insensitive to shRNA knockdown ofTrpm6orTrpm7, however, and was not affected by manipulations of Mg2+, which normally perturbs TRPM6 and TRPM7. To definitively examine the role of these two channels in transduction, we showed that deletion of either or both of their genes selectively in hair cells had no effect on auditory function. We suggest that TRPV5, TRPV6, TRPM6, and TRPM7 are unlikely to be the pore-forming subunit of the hair-cell transduction channel.

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Cross-links between stereocilia in the human organ of Corti

The Journal of Laryngology & Otology ◽

10.1017/s0022215100096237 ◽

1985 ◽

Vol 99 (1) ◽

pp. 11-20 ◽

Cited By ~ 31

Author(s):

Peter H. Rhys Evans ◽

Spiro D. Comis ◽

Michael P. Osborne ◽

James O. Pickles ◽

David J. R. Jeffries

Keyword(s):

Guinea Pig ◽

Hair Cell ◽

Organ Of Corti ◽

Sensory Transduction ◽

Human Organ ◽

Cross Links

AbstractHuman cochleae were fixed in glutaraldehyde, without the use of osmium. Crosslinks were seen between the stereocilia, similar to those we have previously reported for the guinea pig: first, stereocilia of the same row on each hair cell were joined by horizontally-running links; secondly, the shorter stereocilia had pointed tips, each giving rise to a single, vertically-pointing link, which ran upwards to join the adjacent taller stereocilium of the next row. We suggest that distortion of this link is involved in sensory transduction. The links were sparser than had been seen in the guinea pig which may be a reflection of the vulnerability of the links to nonoptimal fixation, and the greater difficulty in producing good fixation in human specimens.

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Tonotopic Gradient in the Developmental Acquisition of Sensory Transduction in Outer Hair Cells of the Mouse Cochlea

Journal of Neurophysiology ◽

10.1152/jn.00136.2009 ◽

2009 ◽

Vol 101 (6) ◽

pp. 2961-2973 ◽

Cited By ~ 94

Author(s):

Andrea Lelli ◽

Yukako Asai ◽

Andrew Forge ◽

Jeffrey R. Holt ◽

Gwenaëlle S. G. Géléoc

Keyword(s):

Hair Cell ◽

Mrna Expression ◽

Hair Cells ◽

Time Course ◽

Expression Patterns ◽

Essential Elements ◽

Outer Hair Cells ◽

Sensory Transduction ◽

Spatiotemporal Expression ◽

Electrophysiological Recordings

Inner ear hair cells are exquisite mechanosensors that transduce nanometer scale deflections of their sensory hair bundles into electrical signals. Several essential elements must be precisely assembled during development to confer the unique structure and function of the mechanotransduction apparatus. Here we investigated the functional development of the transduction complex in outer hair cells along the length of mouse cochlea acutely excised between embryonic day 17 (E17) and postnatal day 8 (P8). We charted development of the stereociliary bundle using scanning electron microscopy; FM1-43 uptake, which permeates hair cell transduction channels, mechanotransduction currents evoked by rapid hair bundle deflections, and mRNA expression of possible components of the transduction complex. We demonstrated that uptake of FM1-43 first occurred in the basal portion of the cochlea at P0 and progressed toward the apex over the subsequent week. Electrophysiological recordings obtained from 234 outer hair cells between E17 and P8 from four cochlear regions revealed a correlation between the pattern of FM1-43 uptake and the acquisition of mechanotransduction. We found a spatiotemporal gradient in the properties of transduction including onset, amplitude, operating range, time course, and extent of adaptation. We used quantitative RT–PCR to examine relative mRNA expression of several hair cell myosins and candidate tip-link molecules. We found spatiotemporal expression patterns for mRNA that encodes cadherin 23, protocadherin 15, myosins 3a, 7a, 15a, and PMCA2 that preceded the acquisition of transduction. The spatiotemporal expression patterns of myosin 1c and PMCA2 mRNA were correlated with developmental changes in several properties of mechanotransduction.

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CNGA3 is expressed in inner ear hair cells and binds to an intracellular C-terminus domain of EMILIN1

Biochemical Journal ◽

10.1042/bj20111255 ◽

2012 ◽

Vol 443 (2) ◽

pp. 463-476 ◽

Cited By ~ 15

Author(s):

Dakshnamurthy Selvakumar ◽

Marian J. Drescher ◽

Jayme R. Dowdall ◽

Khalid M. Khan ◽

James S. Hatfield ◽

...

Keyword(s):

Hair Cell ◽

Hair Cells ◽

Transmembrane Domain ◽

Cell Model ◽

Organ Of Corti ◽

Outer Hair Cells ◽

Sensory Transduction ◽

Molecular Characteristics ◽

Cone Photoreceptors ◽

C Terminus

The molecular characteristics of CNG (cyclic nucleotide-gated) channels in auditory/vestibular hair cells are largely unknown, unlike those of CNG mediating sensory transduction in vision and olfaction. In the present study we report the full-length sequence for three CNGA3 variants in a hair cell preparation from the trout saccule with high identity to CNGA3 in olfactory receptor neurons/cone photoreceptors. A custom antibody targeting the N-terminal sequence immunolocalized CNGA3 to the stereocilia and subcuticular plate region of saccular hair cells. The cytoplasmic C-terminus of CNGA3 was found by yeast two-hybrid analysis to bind the C-terminus of EMILIN1 (elastin microfibril interface-located protein 1) in both the vestibular hair cell model and rat organ of Corti. Specific binding between CNGA3 and EMILIN1 was confirmed with surface plasmon resonance analysis, predicting dependence on Ca2+ with Kd=1.6×10−6 M for trout hair cell proteins and Kd=2.7×10−7 M for organ of Corti proteins at 68 μM Ca2+. Pull-down assays indicated that the binding to organ of Corti CNGA3 was attributable to the EMILIN1 intracellular sequence that follows a predicted transmembrane domain in the C-terminus. Saccular hair cells also express the transcript for PDE6C (phosphodiesterase 6C), which in cone photoreceptors regulates the degradation of cGMP used to gate CNGA3 in phototransduction. Taken together, the evidence supports the existence in saccular hair cells of a molecular pathway linking CNGA3, its binding partner EMILIN1 (and β1 integrin) and cGMP-specific PDE6C, which is potentially replicated in cochlear outer hair cells, given stereociliary immunolocalizations of CNGA3, EMILIN1 and PDE6C.

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