Tmc1 and Tmc2 are essential pore-forming subunits of mechanosensory transduction channels localized to the tips of stereovilli in auditory and vestibular hair cells of the inner ear. To investigate expression and function of Tmc1 and Tmc2 in vestibular organs, we used quantitative polymerase chain reaction (qPCR), fluorescence in situ hybridization - hairpin chain reaction (FISH-HCR), immunostaining, FM1-43 uptake and we measured vestibular evoked potentials (VsEPs) and vestibular ocular reflexes (VORs). We found that Tmc1 and Tmc2 showed dynamic developmental changes, differences in regional expression patterns, and overall expression levels which differed between the utricle and saccule. These underlying changes contributed to unanticipated phenotypic loss of VsEPs and VORs in Tmc1 KO mice. In contrast, Tmc2 KO mice retained VsEPs despite the loss of the calcium buffering protein calretinin, a characteristic biomarker of mature striolar calyx-only afferents. Lastly, we found that neonatal Tmc1 gene replacement therapy is sufficient to restore VsEP in Tmc1 KO mice for up to six months post-injection.

During auditory transduction, sound-evoked vibrations of the hair cell stereociliary bundles open mechanotransducer (MET) ion channels via tip links extending from one stereocilium to its neighbor. How tension in the tip link is delivered to the channel is not fully understood. The MET channel comprises a pore-forming subunit, transmembrane channel-like protein (TMC1 or TMC2), aided by several accessory proteins, including LHFPL5 (lipoma HMGIC fusion partner-like 5). We investigated the role of LHFPL5 in transduction by comparing MET channel activation in outer hair cells of Lhfpl5-/- knockout mice with those in Lhfpl5+/- heterozygotes. The 10 to 90 percent working range of transduction in Tmc1+/+; Lhfpl5+/- was 52 nm, from which the single-channel gating force, Z, was evaluated as 0.34 pN. However, in Tmc1+/+; Lhfpl5-/- mice, the working range increased to 123 nm and Z more than halved to 0.13 pN, indicating reduced sensitivity. Tip link tension is thought to activate the channel via a gating spring, whose stiffness is inferred from the stiffness change on tip link destruction. The gating stiffness was ~40 percent of the total bundle stiffness in wild type but was virtually abolished in Lhfpl5-/-, implicating LHFPL5 as a principal component of the gating spring. The mutation Tmc1 p.D569N reduced the LHFPL5 immunolabeling in the stereocilia and like Lhfpl5-/- doubled the MET working range, but other deafness mutations had no effect on the dynamic range. We conclude that tip-link tension is transmitted to the channel primarily via LHFPL5; residual activation without LHFPL5 may occur by direct interaction between PCDH15 and TMC1.

TMC1 is one of the most common deafness genes causing DFNA36. Patient-derived human induced pluripotent stem cells (iPSCs) provide an opportunity to modelling diseases. TMC1 p.M418K mutation in human is orthologous to Beethoven mice. Here, we investigated the differentiation, morphology and electrophysiological properties of hair cell-like cells (HC-like cells) derived from DFNA36 patient.
Inner ear HC-like cells were induced from iPSCs derived from DFNA36 (TMC1 p.M418K) patient (M+/-), normal control (M+/+) and genetic corrected iPSCs (M+/C). Immunofluorescence, scanning electron microscopy and whole-cell patch-clamp were used to study the mechanism and influence of TMC1 p.M418K mutation.
In this study we successfully generated HC-like cells from iPSCs with three different genotypes. HC-like cells from M+/- showed defected morphology of microvilli and physiological properties compared to M+/+. HC-like cells from M+/C showed recovery in morphology of microvilli and physiological properties.
Our results indicate that TMC1 p.M418K mutation didn\'t influence inner ear hair cell differentiation but the morphology of microvilli and electrophysiological properties and gene correction induced recovery. CRISPR/Cas9 gene therapy is feasible in human patient with TMC1 p.M418K mutation.

TMC1, which encodes transmembrane channel-like protein 1, forms the mechanoelectrical transduction (MET) channel in auditory hair cells, necessary for auditory function. TMC1 variants are known to cause autosomal dominant (DFNA36) and autosomal recessive (DFNB7/11) non-syndromic hearing loss, but only a handful of TMC1 variants underlying DFNA36 have been reported, hampering analysis of genotype-phenotype correlations.
In this study, we retrospectively reviewed 338 probands in an in-house database of genetic hearing loss, evaluating the clinical phenotypes and genotypes of novel TMC1 variants associated with DFNA36. To analyze the structural impact of these variants, we generated two structural models of human TMC1, utilizing the Cryo-EM structure of C. elegans TMC1 as a template and AlphaFold protein structure database. Specifically, the lipid bilayer-embedded protein database was used to construct membrane-embedded models of TMC1. We then examined the effect of TMC1 variants on intramolecular interactions and predicted their potential pathogenicity.
We identified two novel TMC1 variants related to DFNA36 (c.1256T > C:p.Phe419Ser and c.1444T > C:p.Trp482Arg). The affected subjects had bilateral, moderate, late-onset, progressive sensorineural hearing loss with a down-sloping configuration. The Phe419 residue located in the transmembrane domain 4 of TMC1 faces outward towards the channel pore and is in close proximity to the hydrophobic tail of the lipid bilayer. The non-polar-to-polar variant (p.Phe419Ser) alters the hydrophobicity in the membrane, compromising protein-lipid interactions. On the other hand, the Trp482 residue located in the extracellular linker region between transmembrane domains 5 and 6 is anchored to the membrane interfaces via its aromatic rings, mediating several molecular interactions that stabilize the structure of TMC1. This type of aromatic ring-based anchoring is also observed in homologous transmembrane proteins such as OSCA1.2. Conversely, the substitution of Trp with Arg (Trp482Arg) disrupts the cation-π interaction with phospholipids located in the outer leaflet of the phospholipid bilayer, destabilizing protein-lipid interactions. Additionally, Trp482Arg collapses the CH-π interaction between Trp482 and Pro511, possibly reducing the overall stability of the protein. In parallel with the molecular modeling, the two mutants degraded significantly faster compared to the wild-type protein, compromising protein stability.
This results expand the genetic spectrum of disease-causing TMC1 variants related to DFNA36 and provide insight into TMC1 transmembrane protein-lipid interactions.

Ciliated sensory cells such as photo- and olfactory receptors employ multiple types of opsins or hundreds of unique olfactory G-protein coupled receptors to respond to various wavelengths of light or odorants. With respect to hearing and balance, the mechanotransduction machinery involves fewer variants; however, emerging evidence suggests that specialization occurs at the molecular level. To address how the mechanotransduction complex varies in the inner ear, we characterized the expression of paralogous genes that encode components required for mechanotransduction in zebrafish hair cells using RNA-FISH and bioinformatic analysis. Our data indicate striking zonal differences in the expression of two components of the mechanotransduction complex which are known to physically interact, the transmembrane channel-like 1 and 2 (tmc1/2) family members and the calcium and integrin binding 2 and 3 (cib2/3) paralogues. tmc1, tmc2b, and cib3 are largely expressed in peripheral or extrastriolar hair cells, whereas tmc2a and cib2 are enriched in central or striolar hair cells. In addition, a gene implicated in deaf-blindness, ush1c, is highly enriched in a subset of extrastriolar hair cells. These results indicate that specific combinations of these components may optimize responses to mechanical stimuli in subtypes of sensory receptors within the inner ear.

Information about dynamic head motion is conveyed by a central \"striolar\" zone of vestibular hair cells and afferent neurons in the inner ear. How vestibular hair cells are tuned to transduce dynamic stimuli at the molecular level is not well understood. Here we take advantage of the differential expression pattern of tmc1, tmc2a, and tmc2b, which encode channel subunits of the mechanotransduction complex in zebrafish vestibular hair cells. To test the role of various combinations of Tmc subunits in transducing dynamic head movements, we measured reflexive eye movements induced by high-frequency stimuli in single versus double tmc mutants. We found that Tmc2a function correlates with the broadest range of frequency sensitivity, whereas Tmc2b mainly contributes to lower-frequency responses. Tmc1, which is largely excluded from the striolar zone, plays a minor role in sensing lower-frequency stimuli. Our study suggests that the Tmc subunits impart functional differences to the mechanotransduction of dynamic stimuli.Significance Statement Information about dynamic head movements is transmitted by sensory receptors, known as hair cells, in the labyrinth of the inner ear. The sensitivity of hair cells to fast or slow movements of the head differs according to cell type. Whether the mechanotransduction complex that converts mechanical stimuli into electrical signals in hair cells participates in conveying frequency information is not clear. Here we find that the transmembrane channel-like 1/2 genes, which encode a central component of the complex, are differentially expressed in the utricle and contribute to frequency sensitivity in zebrafish.

Calcium and integrin-binding protein 2 (CIB2) and CIB3 bind to transmembrane channel-like 1 (TMC1) and TMC2, the pore-forming subunits of the inner-ear mechano-electrical transduction (MET) apparatus. These interactions have been proposed to be functionally relevant across mechanosensory organs and vertebrate species. Here we show that both CIB2 and CIB3 can form heteromeric complexes with TMC1 and TMC2 and are integral for MET function in mouse cochlea and vestibular end organs as well as in zebrafish inner ear and lateral line. Our AlphaFold 2 models suggest that vertebrate CIB proteins can simultaneously interact with at least two cytoplasmic domains of TMC1 and TMC2 as validated using nuclear magnetic resonance spectroscopy of TMC1 fragments interacting with CIB2 and CIB3. Molecular dynamics simulations of TMC1/2 complexes with CIB2/3 predict that TMCs are structurally stabilized by CIB proteins to form cation channels. Overall, our work demonstrates that intact CIB2/3 and TMC1/2 complexes are integral to hair-cell MET function in vertebrate mechanosensory epithelia.

The mechanoelectrical transduction (MET) channel of cochlear hair cells is gated by the tip link, but the mechanisms that establish the exquisite force sensitivity of this MET channel are not known. Here, we show that the tetraspan lipoma HMGIC fusion partner-like 5 (LHFPL5) directly couples the tip link to the MET channel. Disruption of these interactions severely perturbs MET. Notably, the N-terminal cytoplasmic domain of LHFPL5 binds to an amphipathic helix in TMC1, a critical gating domain conserved between different MET channels. Mutations in the amphipathic helix of TMC1 or in the N-terminus of LHFPL5 that perturb interactions of LHFPL5 with the amphipathic helix affect channel responses to mechanical force. We conclude that LHFPL5 couples the tip link to the MET channel and that channel gating depends on a structural element in TMC1 that is evolutionarily conserved between MET channels. Overall, our findings support a tether model for transduction channel gating by the tip link.

Hearing loss (HL) is the most prevalent sensory disorder whose etiology comes from environmental and/or genetic factors. Approximately 60 % of HL cases are due to mutations in genes responsible for maintaining a normal hearing function. Despite the monogenic inheritance of hereditary hearing loss (HHL), its diagnosis is challenging as both clinical and genetic heterogeneity characterizes it. Through the development of next-generation sequencing (NGS) techniques, the number of identified mutations responsible for HHL has increased exponentially during the last decade. Mutations in the TMC1 have been reported in several patients with nonsyndromic hereditary hearing loss (NSHHL), more precisely in cases with an autosomal recessive inheritance pattern. In this study, we conducted whole-exome sequencing (WES) analysis of a United Arabs Emirates (UAE) family with autosomal recessive nonsyndromic hearing loss (ARNSHL). This analysis revealed segregation of the TMC1 missense mutation c.596A > T (p.Asn199Ile) with the disease. Bioinformatics analysis supported the pathogenic effect of this mutation and predicted its impact at the proteomics level. Molecular docking analysis of TMC2WT, TMC2R123K, TMC2Q205R, and TMC2R123K + Q205R. Finally, protein docking results suggest a role for TMC2 variants in the phenotypic variability observed within the investigated family.

BACKGROUND: Nonsyndromic autosomal recessive hearing loss (DFNB) is an etiologically heterogeneous disorder group showing a wide spectrum of onset ages and severity. DFNB genes are very diverse in their types and functions, making molecular diagnosis difficult. DFNB is particularly frequent in Pakistan, which may be partly due to consanguinity.
OBJECTIVE: This study was performed to determine the genetic causes in Pakistani DFNB families with prelingual onset and to establish genotype-phenotype correlation.
METHODS: Whole exome sequencing and subsequent genetic analysis were performed for 11 Pakistani DFNB families including eight consanguineous families.
RESULTS: We identified eight pathogenic or likely pathogenic mutations in LOXHD1, GJB2, SLC26A4, MYO15A, and TMC1 from six families. The GJB2 mutations were identified in two families each with compound heterozygous mutations and a homozygous mutation. The compound heterozygous mutations in LOXHD1 ([p.D278Y] + [p.D1219E]) and GJB2 [p.M1?] + [p.G12Vfs*2]) were novel. The four missense or start-loss mutations were located at well conserved residues, and most in silico analysis predicted their pathogenicity. In addition to causative mutations, we found compound heterozygous mutations in PTPRQ as variants of uncertain significance.
CONCLUSIONS: This study identified biallelic mutations as the underlying cause of early onset DFNB in six Pakistani families. This study will be helpful in providing an exact molecular diagnosis and treatment of prelingual onset deafness patients.