How Transmembrane Inner Ear (TMIE) plays role in the auditory system: A mystery to us

Abstract Different cellular mechanisms contribute to the hearing sense, so it is obvious that any disruption in such processes leads to hearing impairment that greatly influences the global economy and quality of life of the patients and their relatives. In the past two decades, transmembrane inner ear (TMIE) protein has received a great deal of research interest because its impairments cause hereditary deafness in humans. This evolutionarily conserved membrane protein contributes to a fundamental complex that plays role in the maintenance and function of the sensory hair cells. Although the critical roles of the TMIE in mechanoelectrical transduction or hearing procedures have been discussed, there are little to no review papers summarizing the roles of the TMIE in the auditory system. In order to fill this gap, herein, we discuss the important roles of this protein in the auditory system including its role in mechanotransduction, olivocochlear synapse, morphology and different signalling pathways; we also review the genotype‐phenotype correlation that can per se show the possible roles of this protein in the auditory system.

DFNB: autosomal recessive; and DFNX: X-linked); the following number indicates the order of gene mapping and/or discovery. 11 The cochlea within the inner ear contains the cells responsible for the perception of sound ( Figure 1A, B). Hearing occurs when hair cells of the inner ear convert the sound-induced vibrations into the nerve impulses that are conveyed to the brain for further interpretation. Hair cells are the mechanosensory cells in auditory and vestibular systems in the inner ears of all the vertebrates; they are also detectable in a functionally homologous way in the lateral line organ of the fish. 12 The appellation of hair cells was due to the hair bundles that are present on its apical surface, which involves the stereocilia that are mechanically sensitive organelles of hair cells in the rows with a staircase-like pattern ( Figure 1C). Any damages to the hair cells cause hearing or balance disorder. 13 The stereocilia are connected by the extracellular filament called the tip links. These structures recognize any surrounding movement through mechanotransduction, a transformation of the mechanical force into electrical signals; this process is essential for the sense of proprioception, hearing, touch and balance. 14 One or two mechanotransduction channels are located in every stereocilium of hair cells. 15 They are located on the surface of shorter stereocilia next to the lower end of tip links 15 (Figure 1D).
Tip links transmit the mechanical power onto the transduction channels that can be opened by the stereocilia deflection, and it therefore allows to enter the small positively charged ions like Ca 2+ and K + from the surrounding endolymph. Finally, the depolarized cells send F I G U R E 1 Schematic illustration of the inner ear, cochlear turn cross-section, stereocilia and mechano-electrical transduction (MET) channel. A, Anatomically, the ear consists of three distinct parts including the outer ear, middle ear, and inner ear. The inner ear has two main parts: the cochlea, which is the hearing portion, and the semicircular canals that are the balance portion. B, A cross-section shows the 2.5 turns containing the cochlea duct. C, In the Corti organ, outer hair cells are arranged in three longitudinal rows, whereas the inner hair-cells in a single one, extending along the whole cochlea. D, Schematic diagram of stereocilia of the hair cell. Cadherin 23 and protocadherin-15 (PCDH15) comprise the tip link, which inserts into the stereocilia membrane at the sites of the upper and lower tip densities. Scaffolding proteins-including Myosin VIIa, Harmonin and Sans-bind to the cytoplasmic domain of Cadherin 23 and anchor the tip link. The 'Upper' and the 'Lower Tip Link Density' regions are shown by yellow highlighted zones. Calmodulin binds to the Ca 2+ and also stereocilin protein that, in turn, attaches two stereocilia. E, A model of the MET complex of hair cells. TMC1/2 dimers (TMC1/2) can interact with PCDH15 dimers and TMIE. TMC1/2 dimers by tension exerted perpendicular to the membrane with extracellular tip-link (PCDH15) tension and TMC1/2 tethered intracellularly to CIB2 (dark blue) and Ankyrin (orange) and the actin cytoskeleton the electrical output to the brain through the eighth cranial nerve (reviewed in Ref. [16]). Deflections of stereocilia to the longest one open the transduction channels, whereas deflections in the opposite direction close them. 14 Numerous studies have been conducted to understand the mechanisms of the hair cell mechanotransduction; for example, the research on the genes associated with the inherited HI has introduced several components of the mechanotransduction machin- Usher syndrome 1G (USH1G) and Calcium and integrin-binding protein 2 (CIB2) ( Figure 1E). The transduction channel may contain additional components that have not been identified yet.
Transmembrane Inner Ear, an evolutionarily conserved protein, is one of the main components of the mechanotransduction complex of hair cells. Loss-of-function mutations in the TMIE cause autosomal recessive deafness-6 (DFNB6; OMIM: 600971). [17][18][19][20] Many studies have been performed to understand the roles of TMIE in the maintenance, maturation and development of the inner ear sensory hair cells, but there is no review article summarizing these; to fill, the present review focuses on the genotype-phenotype correlation, the critical role of the TMIE in sensory hair cells in the auditory process, and also the roles of TMIE in regulating the postsynaptic nicotinic acetylcholine receptor (nAChR) function. The presence of a signal peptide is predicted by the TargetP server 22 with a cleavage site that is located between residues 28 and 29.

| TR AN S MEMB R ANE INNER E AR
Homozygous or compound heterozygous mutations in the TMIE gene are associated with the DFNB6 that is featured by a severeto-profound non-syndromic sensorineural hearing loss along with congenital or prelingual onset ( Figure 2; Table 1).
Transmembrane inner ear is expressed in many human tissues, including cochlear tissues as well. 18,20 The presence of the TMIE in the spiral limbus, spiral ligament, organ of Corti and stria vascularis of the rat was also identified. 23 Shen et al 24 showed the expression of Tmie in the hair cells of the mouse organ of Corti. These studies suggest the important roles of TMIE in the hearing process. In fact,

F I G U R E 2
The TMIE protein structure. TMIE consists of an intracellular N-terminus, two transmembrane parts separated by an extracellular loop and a long-charged intracellular C-terminus. The reported mutations are indicated by the yellow-filled circles. The dagger ( †) shows the affected residue in the counterpart of Sr J mouse. The positively charged amino acids are shown by red circles whereas negatively charged residues are indicated by blue ones. There are several lysine (K) residues in C-terminal that are shown by red-filled circles. Three potential protein kinase phosphorylation sites are among 86-93, 103-105 and 131-133 residues-are shown by asterisks (*). The potential binding sites for the TMC1/2 proteins are among 80-100 amino acid positions. Blue arrows highlight the regions that are the target for phosphatidylinositol 4,5-bisphosphate (PIP2)-residues from 80 to 100 and 122 to 142. The two clusters of lysine (K) residues in the Cterminal are indicated by red circles (from 123 to 131 and 150 to 154 residues). The figure is depicted according to the amino acid sequence and also data provided by Ref. [113] the appellation of 'TMIE' was due to the transmembrane domains and also its potential fundamental roles in the inner ear. 18 In the following, we summarize some important information about TMIE and its role in the auditory system.

| Discovery of TMIE
The discovery of the TMIE extremely benefits from the genetic studies of deafness in humans 17 and animal models including mice 18,25 and zebrafish. 19 In 1962, Deol and Robbins 25 reported a new case of spinner (sr) mouse that manifested deafness, typical head tossing, circling and hyperactivity. Light microscopic investigation of the inner ears of the homozygote spinner mice (sr/sr) showed that the auditory and vestibular impairment was potentially peripheral in origin and no clear defects were observed in the gross inner ear morphogenesis 25 ; however, an irregular apical surface of inner and outer hair cells were evident. 18 Later studies mapped the sr to the distal part of chromosome 9 in mice and then comparative gene mapping investigations introduced a region of conserved synteny between the distal mouse chromosome 9 and human chromosome 3. 26 The presence of deafness locus DFNB6 on the short arm of chromosome 3 was described for the first time in a family from southern India with second-cousin marriage (three out of four siblings were deaf). 27 This evidence suggested the spinner strain as a mouse model for the human non-syndromic hearing loss caused by mutations in DFNB6.
Mitchem et al introduced the widening of the Tmie gene by a 40 kb deletion on the sr allele region. They reported two independent mutations in the Tmie gene causing HI and vestibular impairment of the spinner model. 18 The Tmie gene was thoroughly deleted in the sr allele, whereas in sr J , a C > T substitution changed Arginine residue to a premature stop codon at a position of 96 and both cause the same phenotype ( Figure 2). In sum, these studies have paved the way for the discovery of the TMIE gene.

| Genotype of TMIE
Different mutations in the TMIE gene have been reported in association with severe-to-profound non-syndromic hearing loss ( Table 1).
The first five mutations in the TMIE associated with DFNB6 were documented in 2002. 17 Three of them were missense mutations in the cytoplasmic domain at the C-terminal domain including the p.R81C, p.R84W and p.R92W, all located in the exon 3. 17,27 Two substitutions-p.R81C and p.R84W-were located at highly conserved residues, whereas the p.R92W was reported to reside in a tyrosine kinase phosphorylation region ( Figure 2). 28 The 4th mutation was a   acceptor site of the intron 1 (IVS1-2_98delAGCCCAGinsC) which was found in a Pakistani family. As an autosomal recessive mutation, it removes the acceptor splice site prior to the exon 2, therefore causing exon-skipping that in turn results in losing some residues. 17 The frequency of the TMIE mutations was reported by 1.7% among 168 Pakistani patients, whose GJB2-screening was negative. Also, two new mutations were reported including p.E31G in exon 1, and c.212_2A > C at splice acceptor site of intron 2 that was predicted to result in skipping of the 3rd exon and therefore losing a part of the second transmembrane segment and half of the long C-terminal tail of the protein. 28 p.E31G is located in the extracellular domain in the conserved area of the TMIE. 28 The C-terminal of TMIE is rich in charged residues (41/78 amino acids) consisting of two clusters of Lysine and several Arginine residues ( Figure 2). Beyond that, the C-terminal region has three potential phosphorylation sites. 17 A significant portion of the human gene mutations associated with the HI has been reported in such Arginine residues (Table 1), for example p.R81C, p.R84W and p.R92W impress such evolutionary conserved Arginine residues in human cause deafness, confirmed using animal models 14,36 ( Figure 2). Moreover, the substitution of Arginine in the C-terminus of TMIE, at the position of 96, has been reported as a cause for deafness in the sr J mouse, underscoring the essential role of these Arginine residues in the correct function of this protein. 18 TMIE is proposed as a suitable candidate for connection with other functional proteins in the stereocilia; this ability is attributed to the predicted structure of the TMIE along with the special features of C-terminal. 18,28

| TMIE ANIMAL MODEL S
The localization of the TMIE in the stereocilia of hair cells has been firstly indicated in the rat cochlea, 23,37 but later studies on the Tmie LacZ/+ and Tmie LacZ/LacZ mice (an in-frame insertion of a LacZ transgene into the Tmie) showed that the TMIE is located in the stereocilia of inner and outer hair cells next to the lower part of tip link insertion. 14 In fact, animal models-especially the mouse and zebrafish-provide a valuable resource for studying the inherited human HI and also a helpful system for evaluating the function of the candidate genes. As a result, different proteins have been identified that are necessary for the maturation and developmental process of the human inner ear and may have a contribution with TMIE to auditory system. In the following, we summarize a snippet of information about mouse and zebrafish HI models to grasp the importance of TMIE in auditory system.

| Mice
The sr mouse was the first model for human DFNB6 without auditory brainstem response. 25 Degeneration of the hair cells, which was started at P15 and completed at P40, and the degeneration of the auditory nerve cells were also observed. 18 These studies show that mRn49018 genes. 18,39 Tmie is the only common deleted gene between the cir/cir and sr/sr mice.
Comparing the cir/+ and cir/cir mice on the third day of birth (before hair cell degeneration) showed that both have similar inner ear structure, but the cir/cir mice did not absorb the gentamicin, gentamicin-Texas red conjugate or FM1-43, 40 suggesting that the maturation and mechanotransduction have impaired in the hair cells; the Tmie gene has been proposed to be responsible for these events.
Histological examinations in the cir mice indicated the postnatal degeneration of the cochlear epithelium of Corti and spiral ganglion. 39 The neuroepithelial defects of cir including the irregular and shorter stereocilia bundles started earlier at 10 days and were completed more rapidly at 21 days. 20 Collectively, TMIE has been suggested as a critical factor in the auditory system that participates in normal postnatal maturation and maintenance of stereocilia bundles of sensory hair cells. 17,18,39

| Zebrafish
The Tmie ru1000 is a zebrafish model for human DFNB6. In this model, the tmie gene is mutated in two nucleotides of codon 13 from the exon 1, leading to the truncated protein with only 25 amino acids.
The hair cells of this model could not successfully uptake the fluorophores that normally pass through transduction channels 42,43 and their ear structures miss the microphonic potentials in response to the vibratory stimuli. 19 Pacentine et al 44 showed that the 'Gross morphology' is normal in tmie ru1000 mutant zebrafish, whereas tmie ru1000 reveals subtle differences as short and abnormal hair bundles; these findings are in line with the grossly normal hair-cell morphology observed in Tmie −/− mice. Owing to the defect in hair-cell mechanosensitivity, tmie-deficient zebrafish has been suggested to be somehow deaf. 44 Studies have not been able to measure mechanotransduction currents in Tmie-deficient hair cells. 14,44 Interestingly, exogenous expression of a tmie rescued the functional defects in tmie ru1000 and also Tmie-deficient mice. 14,44 Thus, any damage to the mechanotransduction results from the function of the TMIE protein probably generated the morphological changes in the stereocilia or hair bundles.

| TMIE CONNEC TS TO OTHER MEMB ER S IN MECHANOTR ANS DUC TION
The appropriate function of the mechanotransduction channel per se depends on some collaborations between different proteins at the tips of stereocilia. 45 The Tip link involves two connecting proteins, the CDH23 in the upper and PCDH15 in the lower parts ( Figure 1D).  Figure 1D). Protocadherin-15 interacts with the LHFPL5 by two fragments including a transmembrane domain and a short membrane-proximal fragment of the cytoplasmic site, which is commonly observed among different isoforms. 60 In order to perform a ternary complex, TMIE directly connects to the PCDH15-CD2 and indirectly attached to the LHFPL5 that in turn mediates the connection to PCDH15-CD1 and PCDH15-CD3. 14 Hence, it seems that alternative splicing of the cytoplasmic domain of PCDH15 has a critical role in the specific conformation of the ternary complexes of the PCDH15, LHFPL5 and TMIE. 14 Transmembrane inner ear not only does participate in the ternary complex but also binds to pore-forming components of the transduction channels ( Figure 1E). These can show the possible interaction between different important proteins and TMIE in the auditory system.

| LHFPL5
LHFP-like protein 5 is an essential component for conductance and adaptation characteristics of the transducer channel. 60 Structurally and functionally, this protein has a similarity with the 'Transmembrane AMPAR Regulatory Proteins' that allosterically control the pore properties of glutamate receptors. 60 Mutations in LHFPL5 are associated with human DFNB67 70 and have been also reported to cause vestibular dysfunction and deafness in the mice as a result of severe degeneration of Corti. 71 During the otocyte development, LHFPL5 is located throughout the bundle, but with the onset of hearing or postnatally, it gradually moves to the tips of the shorter stereocilia. LHFPL5 cannot be localized at the tip of stereocilia in the Pcdh15-deficient mice 72 and also is essential for localization of the PCDH15 to the site of mechanoelectrical transduction. 60,73 In the absence of the LHFPL5 in the cochlear hair cells, the current of mechanoelectrical transduction is not completely suppressed, 60 whereas the number of tip links significantly decreased. 60 LHFPL5 is also involved in the correct localization of the TMC1 in the mouse cochlear hair cells, 69 although new evidence demonstrates that the Tmc1 and Tmc2b proteins can localize in an independent of the Lhfpl5 in the stereocilia of the zebrafish hair cells. 74

| CIB2
Calcium and integrin-binding protein 2 is involved in the normal operation of mechanotransduction machinery in the auditory hair cells 75 ( Figure 1E). Although it is not a critical factor for localization of the PCDH15 or TMC1/2, this protein connects to the N-terminal domain of the TMC1. 76 CIB2 contributes to the intracellular Ca 2+ signalling, 77 which is called for the hearing process (reviewed in Ref. [78]). The They proposed the probability of transmitting force to the channel through ankyrin 80 ( Figure 1E). Recently, the role of the different parts of TMIE in the mechanosensitivity of the hair cells has been investigated. 36,44 Transmembrane inner ear connects to TMC1/2 through its Cterminal domain that is located near its plasma membrane. 36 Moreover, TMIE binds to the phosphatidylinositol 4,5-bisphosphate (PIP2) from two parts of the C-terminal cytoplasmic domain (residues of 80-100 and 122-142) (Figure 2). 36  Structural modelling has recommended that the TMC1 has a large cavity next to the protein-lipid interface. 65  connecting to the TMIE. 14,36

| MECHANOTR ANS DUC TION AND MORPHOG ENE S IS
The number of mechanotransduction channels is increased rapidly in cochlear hair cells around the time of birth in mice and rats. 85,86 At all developmental stages, it has been identified that the morphological maturation of the hair bundles is not fully complete, that is mechanotransduction currents are necessary to fulfil the morphogenesis of such hair bundles (reviewed in Ref. [87]). Indeed, a threshold level of transduction current activity may determine whether a stereocilium should either incorporate into the bundle or be resorbed as a microvillus. 87 It seems that calcium entering the cell through MET channels can destabilize calcium-sensitive crosslinks between actin filaments in the microvilli. 87 Generally, inner hair cells of the rodent cochlea have three rows of stereocilia: whereas the first row is tall and thick, the second one is short and thick; besides, the third one is short and thin. As discussed, the genetic investigation of HI has revealed various required genes for hair-bundle morphogenesis, among these, human families with deafness can be used fruitfully as a resource to detect genes that are required for hearing and hair-bundle morphogenesis.
We believe that future investigations can unveil the molecular mechanisms behind the hair-bundle morphogenesis and maybe through accompanying TMIE.

| TMIE AND PIP2 S I G NALLING PATHWAY
Stimuli sensed by hair cells increase tension in the tip links that in turn convert the physical forces into chemical signals using inner cells' Ca 2+ signalling pathways. The increased amount of stereociliary Ca 2+ levels trigger MET channel closure through adaptation-a negativefeedback mechanism containing of a shift of the sensitive range of the MET process. 90 Ca 2+ may also affect the function of MET channels indirectly-for example via adenosine 3ʹ,5ʹ-cyclic monophosphate (cAMP)-because the rise of stereocilia Ca 2+ concentration can promote the Ca 2+ -calmodulin-activated type I adenyl cyclase of the hair cells that in turn is followed by activation of protein kinase A and phosphorylation of relevant targets. The increase in the stereocilia Ca 2+ concentration weakens calmodulin binding to the myosin 1c IQ motifs, which in turn interacts with anionic phospholipids in the membrane such as PIP2. 91,92 Phosphatidylinositol 4,5-bisphosphate is a prominent component of the plasma membrane that can alter the function of ion channels. 93 GPSM2 is a regulator of G protein-coupled receptor signalling that, in turn, stimulates phospholipase C (PLC) and is required for normal hearing. GPSM2 along with its partner-GNAI3-is ex- It has been shown that the C-terminal cytoplasmic TMIE domain involves some positively charged residues that mediate binding to phospholipids, especially PIP2, 36 and also C-terminal TMIE affects its binding to TMC1/2, indicating that some of the previously known PIP2 effects on channel function may be mediated by TMIE. This may result in alternations in channel conductance and ion selectivity, suggesting that this part of TMIE regulates the pore properties of the transducer channel. Interestingly, the depletion of PIP2 from hair cells affects MET; this effect is stronger in p.R82C mutant in comparison with the wild-type models. 97 It still is unclear whether the PIP2 dependence arises from its direct interaction with the channel complex or from an indirect effect on lipid mechanics.

| TMIE IS A SUBUNIT FOR α 9α10 n AChR
Nicotinic Acetyl-Choline Receptor (nAChR) is a nicotinic family of cholinergic receptors and α9α10 nAChR counts as a member of this family that is situated in cochlear and vestibular hair cells. 98 F I G U R E 3 Schematic pathway of PIP2 and its possible contribution to the auditory system. Stimulation of receptors coupled to Gα activates Phospholipase C (PLC), which leads to the release of diacylglycerol (DAG) and IP3. GPSM2 is a regulator of G protein-coupled receptor signalling that, in turn, stimulates phospholipase C (PLC). The C-terminal cytoplasmic TMIE domain contains charged amino acids that mediate binding to phospholipids, including PIP2. DAG remains membrane-associated and activates protein kinase C, whereas IP3 diffused into the cell and stimulates the IP3 receptor in the endoplasmic reticulum (ER), leading to mobilization of intracellular Ca 2+ stores. PKC phosphorylates the targets and therefore induces cellular responses including. The figure is redrawn from Refs [59,114] The α9α10 nAChR participates in synaptic currents that originated from medial olivocochlear neurons. The α9α10 nAChR is among the most calcium selective ligand-gated channels which connect to the calcium-activated SK2 potassium channel in the base of hair cells. 98 New evidence determined that hair cell α9α10 nAChR functional expression is regulated by ligand binding and the coexpression of either TMIE and TMEM132e. 99 TMEM132E is deafness-associated gene. 100 This study introduces TMIE as the α9α10 auxiliary subunit. Moreover, it has been identified that aberrant up-regulation of neonatal α9α10 channel function as well as abnormal persistence of cholinergic innervation and α9α10 synaptic transmission beyond P12 in Tmie mutant mice. 99 In agreement with these results, the presence of TMIE in the cell body of hair cells has been demonstrated previously 36 and mRNA expression of TMIE is also enriched together with SK2, α9 and α10 in outer hair cells. 101,102 All in all, these data confirm that TMIE is an auxiliary subunit that participates in channel gating of α9α10 nAChR ( Figure 4) and provides a mechanism to couple cholinergic innervation to postsynaptic nAChR expression and probably enables drug discovery for auditory disorders associated with these hair cell receptors.

| THE THER APEUTI C PER S PEC TIVE OF TMIE
The medial olivocochlear bundle decreases the gain of the cochlear amplifier through reflexive activation by sound. This system improves sound discrimination, refines tonotopic mapping, and protects against sound-induced HI. 98,99 These specifications are a suitable pharmacological target for acoustic trauma, presbycusis, and tinnitus.
Moreover, the pharmacological potential of α9α10 nAChR has been always noticed. [103][104][105] Some studies showed that when cholinergic activity through α9α10 nAChR is enhanced, it could lead to the protection and even repair of the inner ear sensory epithelium from acoustic trauma damages. 104,105 Furthermore, increasing efferent innervation of inner hair cells was observed in age-related mouse models. 106 Generally, this emerging evidence-introducing deafnessassociated TMIE gene as an encoding subunit of α9α10 nAChR in the medial olivocochlear system-increases a new therapeutic perspective for auditory and vestibular disorders.
Hair cell morphology emerges normally in the Tmie-deficient mice at early postnatal ages, which might provide a therapeutic opportunity for the treatment of TMIE-related sensorineural deafness.
Notably, several other mechanotransduction components such as in Tmc1 Bth/WT mice for up to one-year post-injection, raising hopes to treat this gene-related deafness in patients. 111 Interestingly, in another mouse model for DFNB7/11 recessive deafness with a defect in Tmc1, round window membrane injections of synthetic AAV2/ Anc80L65 encoding Tmc1 resulted in the approximately complete restoration of auditory and vestibular function and morphological rescue. 112 Although there are no clinical trials for TMIE in human or animal models, due to the critical roles of this protein in the MET channel, it seems to be viable in a not-so-distant future.

| CON CLUS I ON AND FUTURE PER S PEC TIVE S
The cochlea and the organ of Corti are fascinating structures in which the mechanoelectrotransduction transpires. Over the last 20 years, remarkable steps have been taken forward to a better understanding of this process. Although it has achieved high goals, some obscure points must be unravelled; for example, many genes have been identified that contribute to the hearing process through encoding intracellular motors, adhesion proteins or even scaffolding proteins in the inner ear. However, it is axiomatic that there are still other genes and pathways that remain to be detected. Nonetheless, with the advent of additional high-tech molecular and genetic tools, for example single-cell transcriptomics, it appears likely that the pace of discovery will increase.
Pioneering genetic and physiological studies have indicated a pivotal role of the TMIE protein in the hearing process. Transmembrane inner ear is a critical component of the mechanotransduction machinery of the hair cells and directly and indirectly contributes to the functional molecular mechanism of maturation, development and maintenance of the hair cells; however, in total little is known about its contribution to the hearing process. In this review, we summarized some important findings to illustrate the molecular mechanisms whereby TMIE plays role in the hearing process. We believe that future studies can remove the veil of ignorance and answer the some obscure aspects, for example it is unclear how epigenetic modulations (eg methylation and acetylation) or environmental interventions affect the hearing process through TMIE or MET complex. We still do not know much about how TMIE contributes to controlling α9/α10 nAChR and also plays roles in the MET complex; or is there any relationship between these two different functions? The viable use of TMIE as a target for gene therapy/replacement yet still remains blanketed in mystery. We believe that foreseeable investigations will shed light on TMIE protein and its contribution to the hearing process. This can in turn provide valuable information about the biological aspects of 'hearing', which will pave the way to utilize it effectively for therapeutic purposes.

ACK N OWLED G EM ENT
We thank the staff of the ENT and Head & Neck Research Center, Tehran, Iran, for their contribution.

CO N FLI C T O F I NTE R E S T
The authors have no conflict of interest to declare.

DATA AVA I L A B I L I T Y S TAT E M E N T
The paper is exempt from Data sharing.