Transmembrane Channel-like Research Articles

A Screen for Gene Paralogies Delineating Evolutionary Branching Order of Early Metazoa.

The evolutionary diversification of animals is one of Earth’s greatest marvels, yet its earliest steps are shrouded in mystery. Animals, the monophyletic clade known as Metazoa, evolved wildly divergent multicellular life strategies featuring ciliated sensory epithelia. In many lineages epithelial sensoria became coupled to increasingly complex nervous systems. Currently, different phylogenetic analyses of single-copy genes support mutually-exclusive possibilities that either Porifera or Ctenophora is sister to all other animals. Resolving this dilemma would advance the ecological and evolutionary understanding of the first animals and the evolution of nervous systems. Here we describe a comparative phylogenetic approach based on gene duplications. We computationally identify and analyze gene families with early metazoan duplications using an approach that mitigates apparent gene loss resulting from the miscalling of paralogs. In the transmembrane channel-like (TMC) family of mechano-transducing channels, we find ancient duplications that define separate clades for Eumetazoa (Placozoa + Cnidaria + Bilateria) vs. Ctenophora, and one duplication that is shared only by Eumetazoa and Porifera. In the Max-like protein X (MLX and MLXIP) family of bHLH-ZIP regulators of metabolism, we find that all major lineages from Eumetazoa and Porifera (sponges) share a duplicated gene pair that is sister to the single-copy gene maintained in Ctenophora. These results suggest a new avenue for deducing deep phylogeny by choosing rather than avoiding ancient gene paralogies.

Identification of TMC1 as a relatively common cause for nonsyndromic hearing loss in the Saudi population.

Hearing loss (HL) is the most common sensory disorder worldwide and genetic factors contribute to approximately half of congenital HL cases. HL is subject to extensive genetic heterogeneity, rendering molecular diagnosis difficult. Mutations of the transmembrane channel-like 1 (TMC1) gene cause hearing defects in humans and mice. The precise function of TMC1 protein in the inner ear is unknown, although it is predicted to be involved in functional maturation of cochlear hair cells. TMC1 mutations result in autosomal recessive (DFNB7/11) and sometimes dominant (DFNA36) nonsyndromic HL. Mutations in TMC1 are responsible for a significant portion of HL, particularly in consanguineous populations. To evaluate the importance of TMC1 mutations in the Saudi population, we used a combination of autozygome-guided candidate gene mutation analysis and targeted next generation sequencing in 366 families with HL previously shown to lack mutations in GJB2. We identified 12 families that carried five causative TMC1 mutations; including three novel (c.362+3A > G; c.758C > T [p.Ser253Phe]; c.1396_1398delACC [p.Asn466del]) and two reported mutations (c.100C > T [p.Arg34Ter]; c.1714G > A [p.Asp572Asn]). Each of the identified recessive mutation was classified as severe, by both age of onset and severity of HL. Similarly, consistent with the previously reported dominant variant p.Asp572Asn, the HL phenotype was progressive. Eight families in our cohort were found to share the pathogenic p.Arg34Ter mutation and linkage disequilibrium was observed between p.Arg34Ter and SNPs investigated. Our results indicate that TMC1 mutations account for about 3.3% (12/366) of Saudi HL cases and that the recurrent TMC1 mutation p.Arg34Ter is likely to be a founder mutation.

TMC1 and TMC2 Proteins Are Pore-Forming Subunits of Mechanosensitive Ion Channels

Transmembrane channel-like (TMC) 1 and 2 are required for the mechanotransduction of mouse inner ear hair cells and localize to the site of mechanotransduction in mouse hair cell stereocilia. However, it remains unclear whether TMC1 and TMC2 are indeed ion channels and whether they can sense mechanical force directly. Here we express TMC1 from the green sea turtle (CmTMC1) and TMC2 from the budgerigar (MuTMC2) in insect cells, purify and reconstitute the proteins, and show that liposome-reconstituted CmTMC1 and MuTMC2 proteins possess ion channel activity. Furthermore, by applying pressure to proteoliposomes, we demonstrate that both CmTMC1 and MuTMC2 proteins can indeed respond to mechanical stimuli. In addition, CmTMC1 mutants corresponding to human hearing loss mutants exhibit reduced or no ion channel activity. Taken together, our results show that the CmTMC1 and MuTMC2 proteins are pore-forming subunits of mechanosensitive ion channels, supporting TMC1 and TMC2 as hair cell transduction channels.

The contribution of TMC1 to adaptation of mechanoelectrical transduction channels in cochlear outer hair cells.

Key points Hair cell mechanoelectrical transducer channels are opened by deflections of the hair bundle about a resting position set by incompletely understood adaptation mechanisms.We used three characteristics to define adaptation in hair cell mutants of transmembrane channel‐like proteins, TMC1 and TMC2, which are considered to be channel constituents.The results obtained demonstrate that the three characteristics are not equivalent, and raise doubts about simple models in which intracellular Ca2+ regulates adaptation.Adaptation is faster and more effective in TMC1‐containing than in TMC2‐containing transducer channels. This result ties adaptation to the channel complex, and suggests that TMC1 is a better isoform for use in cochlear hair cells.We describe a TMC1 point mutation, D569N, that reduces the resting open probability and Ca2+ permeability of the transducer channels, comprising properties that may contribute to the deafness phenotype. Recordings of mechanoelectrical transducer (MET) currents in cochlear hair cells were made in mice with mutations of transmembrane channel‐like (TMC) protein to examine the effects on fast transducer adaptation. Adaptation was faster and more complete in Tmc2–/– than in Tmc1–/–, although this disparity was not explained by differences in Ca2+ permeability or Ca2+ influx between the two isoforms, with TMC2 having the larger permeability. We made a mouse mutation, Tmc1 p.D569N, homologous to a human DFNA36 deafness mutation, which also had MET channels with lower Ca2+‐permeability but showed better fast adaptation than wild‐type Tmc1+/+ channels. Consistent with the more effective adaptation in Tmc1 p.D569N, the resting probability of MET channel opening was smaller. The three TMC variants studied have comparable single‐channel conductances, although the lack of correlation between channel Ca2+ permeability and adaptation opposes the hypothesis that adaptation is controlled simply by Ca2+ influx through the channels. During the first postnatal week of mouse development, the MET currents amplitude grew, and transducer adaptation became faster and more effective. We attribute changes in adaptation partly to a developmental switch from TMC2‐ to TMC1‐ containing channels and partly to an increase in channel expression. More complete and faster adaptation, coupled with larger MET currents, may account for the sole use of TMC1 in the adult cochlear hair cells.

TMC1 is an essential component of a leak channel that modulates tonotopy and excitability of auditory hair cells in mice

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.

An uncharacterized region within the N-terminus of mouse TMC1 precludes trafficking to plasma membrane in a heterologous cell line

Mechanotransduction by hair cell stereocilia lies at the heart of sound detection in vertebrates. Considerable effort has been put forth to identify proteins that comprise the hair cell mechanotransduction apparatus. TMC1, a member of the transmembrane channel-like (TMC) family, was identified as a core protein of the mechanotransduction complex in hair cells. However, the inability of TMC1 to traffic through the endoplasmic reticulum in heterologous cellular systems has hindered efforts to characterize its function and fully identify its role in mechanotransduction. We developed a novel approach that allowed for the detection of uncharacterized protein regions, which preclude trafficking to the plasma membrane (PM) in heterologous cells. Tagging N-terminal fragments of TMC1 with Aquaporin 3 (AQP3) and GFP fusion reporter, which intrinsically label PM in HEK293 cells, indicated that residues at the edges of amino acid sequence 138–168 invoke intracellular localization and/or degradation. This signal is able to preclude surface localization of PM protein AQP3 in HEK293 cells. Substitutions of the residues by alanine or serine corroborated that the information determining the intracellular retention is present within amino acid sequence 138–168 of TMC1 N-terminus. This novel signal may preclude the proper trafficking of TMC1 to the PM in heterologous cells.

Distinct functions of TMC channels: a comparative overview.

In the past two decades, transmembrane channel-like (TMC) proteins have attracted a significant amount of research interest, because mutations of Tmc1 lead to hereditary deafness. As evolutionarily conserved membrane proteins, TMC proteins are widely involved in diverse sensorimotor functions of many species, such as hearing, chemosensation, egg laying, and food texture detection. Interestingly, recent structural and physiological studies suggest that TMC channels may share a similar membrane topology with the Ca2+-activated Cl- channel TMEM16 and the mechanically activated OSCA1.2/TMEM63 channel. Namely, these channels form dimers and each subunit consists of ten transmembrane segments. Despite this important structural insight, a key question remains: what is the gating mechanism of TMC channels? The major technical hurdle to answer this question is that the reconstitution of TMC proteins as functional ion channels has been challenging in mammalian heterologous systems. Since TMC channels are conserved across taxa, genetic studies of TMC channels in model organisms such as C. elegans, Drosophila, and zebrafish may provide us critical information on the physiological function and regulation of TMCs. Here, we present a comparative overview on the diverse functions of TMC channels in different species.

Transmembrane Channel-Like 5 (TMC5) promotes prostate cancer cell proliferation through cell cycle regulation

BackgroundIn this study, we aimed to investigate the biological functions of Transmembrane Channel-Like 5 (TMC5) by bioinformatics and molecular biology methods in prostate cancer (PCa). MethodsWe assessed the mRNA expression level of TMC5 in PCa with public database the Cancer Genome Atlas (TCGA) and Oncomine. The biological functions were demonstrated by bioinformatics methods and siRNA mediated knockdown experiments. Reverse transcription polymerase chain reaction (RT-PCR), immunohistochemical (IHC) experiments and microarray analysis were performed to confirm the results. ResultsTMC5 expression level was significantly up-regulated in 4 independent PCa cohorts compared to normal group. Moreover, TMC5 has higher diagnostic efficiency than PSA-KLK3 (AUC (Area Under Curve) = 0.772, P < 0.001). The high expression of TMC5 was associated with clinical Gleason score, prostate-specific antigen (PSA) level, androgen receptor (AR) activity score and the genes which were known frequently mutated in PCa progression (P < 0.05). Functionally, Gene Otology (GO) analysis suggested that TMC5 was related to cell development; TMC5 knockdown significantly inhibited PCa cells proliferation by arresting cell cycle at G1 phase. Drug sensitivity experiments showed TMC5 knockdown significantly enhanced cells sensitivity to 5-Fluorouracil. Microarray analysis showed TMC5 knockdown significantly inhibited cell cycle and tumor progression. ConclusionOur findings revealed that TMC5 promoted PCa cell proliferation through cell cycle regulation and could be a powerful and hopeful target for PCa treatment.

Sweet neurons inhibit texture discrimination by signaling TMC-expressing mechanosensitive neurons in Drosophila.

Integration of stimuli of different modalities is an important but incompletely understood process during decision making. Here, we show that Drosophila are capable of integrating mechanosensory and chemosensory information of choice options when deciding where to deposit their eggs. Specifically, females switch from preferring the softer option for egg-laying when both options are sugar free to being indifferent between them when both contain sucrose. Such sucrose-induced indifference between options of different hardness requires functional sweet neurons, and, curiously, the Transmembrane Channel-like (TMC)-expressing mechanosensitive neurons that have been previously shown to promote discrimination of substrate hardness during feeding. Further, axons of sweet neurons directly contact axons of TMC-expressing neurons in the brain and stimulation of sweet neurons increases Ca2+ influx into axons of TMC-expressing neurons. These results uncover one mechanism by which Drosophila integrate taste and tactile information when deciding where to deposit their eggs and reveal that TMC-expressing neurons play opposing roles in hardness discrimination in two different decisions.

Improved\xa0TMC1 gene therapy restores hearing and balance in mice with genetic inner ear disorders

Fifty percent of inner ear disorders are caused by genetic mutations. To develop treatments for genetic inner ear disorders, we designed gene replacement therapies using synthetic adeno-associated viral vectors to deliver the coding sequence for Transmembrane Channel-Like (Tmc) 1 or 2 into sensory hair cells of mice with hearing and balance deficits due to mutations in Tmc1 and closely related Tmc2. Here we report restoration of function in inner and outer hair cells, enhanced hair cell survival, restoration of cochlear and vestibular function, restoration of neural responses in auditory cortex and recovery of behavioral responses to auditory and vestibular stimulation. Secondarily, we find that inner ear Tmc gene therapy restores breeding efficiency, litter survival and normal growth rates in mouse models of genetic inner ear dysfunction. Although challenges remain, the data suggest that Tmc gene therapy may be well suited for further development and perhaps translation to clinical application.

Honing In on TMC as the Hair Cell’s Transduction Channel

The identity of the inner ear's transduction channel has bedeviled auditory neuroscientists for decades. In this issue of Neuron, Pan etal. (2018) report the most convincing evidence to date implicating the transmembrane channel-like (TMC) proteins as forming the pore of the transduction channel.

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

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.

Identification of four TMC1 variations in different Chinese families with hereditary hearing loss.

BackgroundVariants in TMC1 (transmembrane channel‐like 1) can cause both autosomal dominant and recessive hearing loss in human population. Mice with Tmc1 variants have been shown to be ideal animal models for gene therapy. In this article, we report four TMC1 variants in four different Chinese families and the follow‐up auditory phenotype of a previously reported family.MethodsFour families with TMC1 variants, as well as a previously described family with TMC1 variant orthologous to the Beethoven mouse, were recruited in this study. A comprehensive auditory evaluation was performed on all ascertained family members. High‐throughput sequencing was conducted using genomic DNA from the probands and other family members to identify probable deafness genes.ResultsWe identified four TMC1 (NM_138691.2) variations, including two pathogenic variants, c.1714G>A, and c.1253T>A, one likely pathogenic variant, c.[797T>C];[797T>C], and one single nucleotide polymorphism (SNP), c.2276G>A. Among these variants, c.[797T>C];[797T>C] is a novel likely pathogenic variant, and c.1714G>A and c.1253T>A are known pathogenic variants at the DFNB7/11 (DFNA36) locus. Phenotype‐genotype correlation analysis of TMC1 variants showed that the TMC1 dominant variation‐related phenotype was late‐onset, progressive, high frequency to all frequency sensorineural hearing loss, while the TMC1 recessive variant was related to congenital all frequency sensorineural hearing impairment.ConclusionsTwo pathogenic, one likely pathogenic variants and one SNP of TMC1 were identified in four Chinese families with hereditary hearing loss, indicating that TMC1 may be a more frequent cause of hearing loss than expected. TMC1 variants related to hearing loss result in specific phenotypes. The TMC1 c.1253T>A (p.M418K) variation, homologous to the Tmc1 c. 1235 T> A (p.M412K) variant in Beethoven mice, was the second report of this variant in human patients with hearing loss, suggesting the possibility to translational gene therapy from Beethoven mice to human patients.

Bioinformatic characterization of the Anoctamin Superfamily of Ca2+-activated ion channels and lipid scramblases

Our laboratory has developed bioinformatic strategies for identifying distant phylogenetic relationships and characterizing families and superfamilies of transport proteins. Results using these tools suggest that the Anoctamin Superfamily of cation and anion channels, as well as lipid scramblases, includes three functionally characterized families: the Anoctamin (ANO), Transmembrane Channel (TMC) and Ca2+-permeable Stress-gated Cation Channel (CSC) families; as well as four families of functionally uncharacterized proteins, which we refer to as the Anoctamin-like (ANO-L), Transmembrane Channel-like (TMC-L), and CSC-like (CSC-L1 and CSC-L2) families. We have constructed protein clusters and trees showing the relative relationships among the seven families. Topological analyses suggest that the members of these families have essentially the same topologies. Comparative examination of these homologous families provides insight into possible mechanisms of action, indicates the currently recognized organismal distributions of these proteins, and suggests drug design potential for the disease-related channel proteins.

TMC Proteins Modulate Egg Laying and Membrane Excitability through a Background Leak Conductance in C. elegans

Membrane excitability is a fundamentally important feature for all excitable cells including both neurons and muscle cells. However, the background depolarizing conductances in excitable cells, especially in muscle cells, are not well characterized. Although mutations in transmembrane channel-like (TMC) proteins TMC1 and TMC2 cause deafness and vestibular defects in mammals, their precise action modes areelusive. Here, we discover that both TMC-1 and TMC-2 are required for normal egg laying in C.elegans. Mutations in these TMC proteins cause membrane hyperpolarization and disrupt the rhythmic calcium activities in both neurons and muscles involved in egg laying. Mechanistically, TMC proteins enhance membrane depolarization through background leak currents and ectopic expression of both C.elegans and mammalian TMC proteins results in membrane depolarization. Therefore, we have identified an unexpected role of TMC proteins in modulating membrane excitability. Our results may provide mechanistic insights into the functions of TMC proteins in hearing loss and other diseases.

Identification of a novel homozygous mutation in transmembrane channel like 1 (TMC1) gene, one of the second-tier hearing loss genes after GJB2 in India.

Background & objectives:Hearing impairment is a common and heterogeneous sensory disorder in humans. Among about 90 genes, which are known to be associated with hearing impairment, mutations in the GJB2 (gap junction protein beta 2) gene are the most prevalent in individuals with hereditary hearing loss. Contribution of the other deafness-causing genes is relatively poorly understood. Here, we present our findings on two families with transmembrane channel like 1 (TMC1) gene variants of the 47 families with nonsyndromic hearing loss (NSHL) studied.Methods:Forty seven families including 26 consanguineous families with at least two hearing impaired children and one normal hearing child and 21 non-consanguineous families having at least three hearing impaired children and one normal hearing child were enrolled for this study. Genetic linkage studies were carried out in 41 families that were GJB2 (Connexin 26) negative. Seven polymorphic short tandem repeat markers at the DFNB7/11 locus were studied employing fluorescently labelled markers.Results:A novel homozygous missense mutation c.1283C>A (p.Ala428Asp) was identified co-segregating with hearing loss. This change results in substitution of a highly conserved polar alanine to a charged aspartic acid and is predicted to be deleterious. In addition, a previously reported nonsense mutation, p.R34X in TMC1, was found.Interpretation & conclusions:While mutations in TMC1 are not as common a cause of NSHL as those in GJB2, TMC1 should be considered for diagnostic investigations in cases of NSHL in GJB2-negative families.

The Basis of Food Texture Sensation in Drosophila

Food texture has enormous effects on food preferences. However, the mechanosensory cells and key molecules responsible for sensing the physical properties of food are unknown. Here, we show that akin to mammals, the fruit fly, Drosophila melanogaster, prefers food with a specific hardness or viscosity. This food texture discrimination depends upon a previously unknown multidendritic (md-L) neuron, which extends elaborate dendritic arbors innervating the bases of taste hairs. The md-L neurons exhibit directional selectivity in response to mechanical stimuli. Moreover, these neurons orchestrate different feeding behaviors depending on the magnitude of the stimulus. We demonstrate that the single Drosophila transmembrane channel-like (TMC) protein is expressed in md-L neurons, where it is required for sensing two key textural features of food-hardness and viscosity. We propose that md-L neurons are long sought after mechanoreceptor cells through which food mechanics are perceived and encoded by a taste organ, and that this sensation depends on TMC. VIDEO ABSTRACT.

Sensing pH with TMCs

Transmembrane channel-like (TMC) proteins have been implicated in hair cell mechanotransduction, Drosophila proprioception, and sodium sensing in the nematode C.elegans. In this issue of Neuron, Wang etal. (2016) report that C.elegans TMC-1 mediates nociceptor responses to high pH, not sodium, allowing the nematode to avoid strongly alkaline environments in which most animals cannot survive.

Mutation Screening of Exons 7 and 13 of the TMC1 Gene in Autosomal Recessive Non-syndromic Hearing Loss (ARNSHL) in Iran.

Background:Non-syndromic hearing loss (NSHL) is the most common birth defect and occurs in approximately 1/1,000 newborns. NSHL is a heterogeneous trait and can arise due to both genetic and environmental factors. Mutations of the transmembrane channel-like 1 (TMC1) gene cause non-syndromic deafness in humans and mice.Objectives:The aim of the present study was to investigate the association of TMC1 gene mutations of the locus DFNB7/11 in exons 7 and 13 in a cohort of 100 patients with hearing loss in Iran using polymerase chain reaction–single-stranded conformation polymorphism (PCR-SSCP), heteroduplex analysis (HA), and DNA sequencing.Patients and Methods:In this experimental study, the blood samples of 100 NSHL patients were collected from 10 provinces in Iran. These patients had a mean age of 16.5 ± 2.01 years and 74.15% of their parents had consanguinity. DNA was extracted from specimens and mutations of exons 7 and 13 of the TMC1 gene were investigated using PCR-SSCP. All samples were checked via HA reaction and suspected specimens with shift bands were subjected to DNA sequencing for investigation of any gene variation.Results:In this study, no mutation was found in the two exons of TMC1 gene. It was concluded from these results that mutations of the TMC1 gene’s special exons 7 and 13 have a low contribution in patients and are not great of clinical importance in these Iranian provinces.Conclusions:More studies are needed to investigate the relationship between other parts of this gene with hearing loss in different populations through the country. More research could clarify the role of this gene and its relation with deafness and provide essential information for the prevention and management of auditory disorders caused by genetic factors in the Iranian population.

Exome sequencing identifies a mutation in TMC1 as a novel cause of autosomal recessive nonsyndromic hearing loss.

BackgroundAutosomal recessive non-syndromic hearing loss (ARNSHL) is highly heterogeneous, and mutations in the gene encoding transmembrane channel-like 1 (TMC1) have been implicated in its development. To date, 35 homozygous mutations in TMC1, identified in over 60 families worldwide, have been shown to be associated with ARNSHL. However, few of these mutations were detected in the Chinese population. In this study, we describe a pathogenic missense mutation located in the T5–T6 domain of TMC1 in a three-generation Chinese family with 14 members.MethodsWhole exome sequencing was performed using samples from one unaffected individual and two affected individuals to systematically search for deafness susceptibility genes. Candidate mutations and cosegregation of the phenotype were verified by polymerase chain reaction and Sanger sequencing in all of the family members.ResultsWe identified a novel TMC1 mutation in exon 20, c.1979C>T, p.P660L, which segregated with prelingual autosomal recessive sensorineural hearing loss.ConclusionsWe found a new missense mutation in the T5–T6 domain of TMC1, which is highly conserved in many species. These data support the potential conserved role of p.P660L in human TMC1 function.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-016-0780-5) contains supplementary material, which is available to authorized users.