Tbx2 is a master regulator of inner versus outer hair cell differentiation.

The cochlea uses two types of mechanosensory cell to detect sounds. A single row of inner hair cells (IHCs) synapse onto neurons to transmit sensory information to the brain, and three rows of outer hair cells (OHCs) selectively amplify auditory inputs1. So far, two transcription factors have been implicated in the specific differentiation of OHCs, whereas, to our knowledge, none has been identified in the differentiation of IHCs2-4. One such transcription factor for OHCs, INSM1, acts during a crucial embryonic period to consolidate the OHC fate, preventing OHCs from transdifferentiating into IHCs2. In the absence of INSM1, embryonic OHCs misexpress a core set of IHC-specific genes, which we predict are involved in IHC differentiation. Here we find that one of these genes, Tbx2, is a master regulator of IHC versus OHC differentiation in mice. Ablation of Tbx2 in embryonic IHCs results in their development as OHCs, expressing early OHC markers such as Insm1 and eventually becoming completely mature OHCs in the position of IHCs. Furthermore, Tbx2 is epistatic to Insm1: in the absence of both genes, cochleae generate only OHCs, which suggests that TBX2 is necessary for the abnormal transdifferentiation of INSM1-deficient OHCs into IHCs, as well as for normal IHC differentiation. Ablation of Tbx2 in postnatal, largely differentiated IHCs makes them transdifferentiate directly into OHCs, replacing IHC features with those of mature and not embryonic OHCs. Finally, ectopic expression of Tbx2 in OHCs results in their transdifferentiation into IHCs. Hence, Tbx2 is both necessary and sufficient to make IHCs distinct from OHCs and maintain this difference throughout development.

Author Correction: Trans-differentiation of outer hair cells into inner hair cells in the absence of INSM1.

In Figs. 1e and 2g of this Letter, the labels 'actin' and 'VGLUT3', respectively, should have been in red instead of green font. This has been corrected online.

Trans-differentiation of outer hair cells into inner hair cells in the absence of INSM1.

The mammalian cochlea contains two types of mechanosensory hair cell that have different and critical functions in hearing. Inner hair cells (IHCs), which have an elaborate presynaptic apparatus, signal to cochlear neurons and communicate sound information to the brain. Outer hair cells (OHCs) mechanically amplify sound-induced vibrations, providing enhanced sensitivity to sound and sharp tuning. Cochlear hair cells are solely generated during development, and hair cell death-most often of OHCs-is the most common cause of deafness. OHCs and IHCs, together with supporting cells, originate in embryos from the prosensory region of the otocyst, but how hair cells differentiate into two different types is unknown1-3. Here we show that Insm1, which encodes a zinc finger protein that is transiently expressed in nascent OHCs, consolidates their fate by preventing trans-differentiation into IHCs. In the absence of INSM1, many hair cells that are born as OHCs switch fates to become mature IHCs. To identify the genetic mechanisms by which Insm1 operates, we compared the transcriptomes of immature IHCs and OHCs, and of OHCs with and without INSM1. In OHCs that lack INSM1, a set of genes is upregulated, most of which are normally preferentially expressed by IHCs. The homeotic cell transformation of OHCs without INSM1 into IHCs reveals a mechanism by which these neighbouring mechanosensory cells begin to differ: INSM1 represses a core set of early IHC-enriched genes in embryonic OHCs and makes them unresponsive to an IHC-inducing gradient, so that they proceed to mature as OHCs. Without INSM1, some of the OHCs in which these few IHC-enriched transcripts are upregulated trans-differentiate into IHCs, identifying candidate genes for IHC-specific differentiation.

Codeficiency of Lysosomal Mucolipins 3 and 1 in Cochlear Hair Cells Diminishes Outer Hair Cell Longevity and Accelerates Age-Related Hearing Loss.

Acquired hearing loss is the predominant neurodegenerative condition associated with aging in humans. Although mutations on several genes are known to cause congenital deafness in newborns, few genes have been implicated in age-related hearing loss (ARHL), perhaps because its cause is likely polygenic. Here, we generated mice lacking lysosomal calcium channel mucolipins 3 and 1 and discovered that both male and female mice suffered a polygenic form of hearing loss. Whereas mucolipin 1 is ubiquitously expressed in all cells, mucolipin 3 is expressed in a small subset of cochlear cells, hair cells (HCs) and marginal cells of the stria vascularis, and very few other cell types. Mice lacking both mucolipins 3 and 1, but not either one alone, experienced hearing loss as early as at 1 month of age. The severity of hearing impairment progressed from high to low frequencies and increased with age. Early onset of ARHL in these mice was accompanied by outer HC (OHC) loss. Adult mice conditionally lacking mucolipins in HCs exhibited comparable auditory phenotypes, thereby revealing that the reason for OHC loss is mucolipin codeficiency in the HCs and not in the stria vascularis. Furthermore, we observed that OHCs lacking mucolipins contained abnormally enlarged lysosomes aggregated at the apical region of the cell, whereas other organelles appeared normal. We also demonstrated that these aberrant lysosomes in OHCs lost their membrane integrity through lysosomal membrane permeabilization, a known cause of cellular toxicity that explains why and how OHCs die, leading to premature ARHL.SIGNIFICANCE STATEMENT Presbycusis, or age-related hearing loss (ARHL), is a common characteristic of aging in mammals. Although many genes have been identified to cause deafness from birth in both humans and mice, only a few are known to associate with progressive ARHL, the most prevalent form of deafness. We have found that mice lacking two lysosomal channels, mucolipins 3 and 1, suffer accelerated ARHL due to auditory outer hair cell degeneration, the most common cause of hearing loss and neurodegenerative condition in humans. Lysosomes lacking mucolipins undergo organelle membrane permeabilization and promote cytotoxicity with age, revealing a novel mechanism of outer hair cell degeneration and ARHL. These results underscore the importance of lysosomes in hair cell survival and the maintenance of hearing.

Mucolipin co-deficiency causes accelerated endolysosomal vacuolation of enterocytes and failure-to-thrive from birth to weaning.

During the suckling period, intestinal enterocytes are richly endowed with endosomes and lysosomes, which they presumably utilize for the uptake and intracellular digestion of milk proteins. By weaning, mature intestinal enterocytes replace those rich in lysosomes. We found that mouse enterocytes before weaning express high levels of two endolysosomal cation channels, mucolipins 3 and 1 -products of Trpml3 and Trpml1 genes; moreover neonatal enterocytes of mice lacking both mucolipins (Trpml3-/-;Trpml1-/-) vacuolated pathologically within hours of birth and remained so until weaning. Ultrastructurally and chemically these fast-forming vacuoles resembled those that systemically appear in epithelial cells of mucolipidosis type IV (MLIV) patients, which bear mutations in Trpml1. Hence, lack of both mucolipins 1 and 3 causes an accelerated MLIV-type of vacuolation in enterocytes. The vacuoles were aberrant hybrid organelles with both endosomal and lysosomal components, and were not generated by alterations in endocytosis or exocytosis, but likely by an imbalance between fusion of lysosomes and endosomes and their subsequent scission. However, upon extensive vacuolation enterocytes displayed reduced endocytosis from the intestinal lumen, a defect expected to compromise nutrient uptake. Mice lacking both mucolipins suffered a growth delay that began after birth and continued through the suckling period but recovered after weaning, coinciding with the developmental period of enterocyte vacuolation. Our results demonstrate genetic redundancy between lysosomal mucolipins 3 and 1 in neonatal enterocytes. Furthermore, our Trpml3-/-;Trpml1-/- mice represent a polygenic animal model of the poorly-understood, and often intractable, neonatal failure-to-thrive with intestinal pathology. Our results implicate lysosomes in neonatal intestinal pathologies, a major cause of infant mortality worldwide, and suggest transient intestinal dysfunction might affect newborns with lysosomal storage disorders. Finally, we conclude that mucolipin-endowed lysosomes in the young play an evolutionarily-conserved role in the intracellular digestion of maternally-provided nutrients, whether milk in mammals or yolk in oviparous species.

TRPML2 and mucolipin evolution.

The TRPML2 protein, encoded by the Mcoln2 gene, is one of the three mucolipins (TRPML1-3), a subset of the TRP superfamily of ion channels. Although there are no thorough studies on the cellular distribution of TRPML2, its mRNA appears to be largely restricted to lymphocytes and other immune cells. This contrasts with the ubiquitous expression of TRPML1 and the limited but diverse expression of TRPML3 and clearly suggests a specialized role for TRPML2 in immunity. Localization studies indicate that TRPML2 is present in lysosomes (including the specialized lysosome-related organelle that B-lymphocytes use for processing of the antigen-bound B-cell receptor), late endosomes, recycling endosomes, and, at a much lower level, the plasma membrane. Heterologously expressed TRPML2, like TRPML1 and/or TRPML3, forms ion channels that can be activated by a gain-of-function mutation (alanine to proline in the fifth transmembrane domain, close to the pore) that favors the open state, by a transient reduction of extracellular sodium followed by sodium replenishment, by small chemicals related to sulfonamides, and by PI(3,5)P2, a rare phosphoinositide that naturally accumulates in the membranes of endosomes and lysosomes and thus could act as a physiologically relevant agonist. TRPML2 channels are inwardly rectifying and permeable to Ca(2+), Na(+), and Fe(2+). When heterologously co-expressed, TRPML2 can form heteromultimers with TRPML1 and TRPML3. In B-lymphocytes, TRPML2 and TRPML1 may play redundant roles in the function of their specialized lysosome. Although the specific subcellular function of TRPML2 is unknown, distribution and channel properties suggest roles in calcium release from endolysosomes, perhaps to regulate vesicle fusion and/or subsequent scission or to release calcium from intracellular acidic stores for signaling in the cytosol. Alternatively, TRPML2 could function in the plasma membrane, and its abundance in vesicles of the endocytic pathway could simply be due to regulation by endocytosis and exocytosis. The Mcoln2 gene is closely downstream from and in the same orientation as Mcoln3 in the genomes of most jawed vertebrates (from humans to sharks) with the exception of pigs, Xenopus tropicalis, and ray-finned fishes. The close homology of TRPML2 and 3 (closer to each other than to TRPML1) suggests that Mcoln2 and Mcoln3 arose from unequal crossing over that duplicated a common ancestor and placed both gene copies in tandem. These genes would have come apart subsequently in pigs, Xenopus, and the ancestor to ray-finned fishes. All jawed vertebrates for which we have thorough genomic knowledge have distinct Mcoln1, 2, and 3 genes (except ray-finned fishes which, probably due to the whole-genome duplication in their common ancestor, have two Mcoln1-like genes and two Mcoln3-like genes, although only one Mcoln2 gene). However, the available genomes of invertebrate deuterostomes (a sea urchin, lancelet, and two tunicates) contain a single mucolipin gene that is equally distant from the three vertebrate mucolipins. Hence, vertebrate mucolipins arose through two rounds of gene duplication (the first one likely producing Mcoln1 and the ancestor to Mcoln2 and 3) at some time between the onset of craniates and that of jawed vertebrates. This is also the evolutionary period during which adaptive immunity appeared. Given the restricted expression of TRPML2 in immune cells, this evolutionary history suggests a functional role in the adaptive immunity characteristic of vertebrates.

Precision patterning: How inner hair cells "hop" to it.

A combination of Notch-mediated lateral inhibition, mechanical forces, and differential adhesion generates a single row of alternating inner hair and supporting cells.

Espin overexpression causes stereocilia defects and provides an anti-capping effect on actin polymerization.

Stereocilia are actin-based projections of hair cells that are arranged in a step like array, in rows of increasing height, and that constitute the mechanosensory organelle used for the senses of hearing and balance. In order to function properly, stereocilia must attain precise sizes in different hair cell types and must coordinately form distinct rows with varying lengths. Espins are actin-bundling proteins that have a well-characterized role in stereocilia formation; loss of function mutations in Espin result in shorter stereocilia and deafness in the jerker mouse. Here we describe the generation of an Espin overexpressing transgenic mouse line that results in longer first row stereocilia and discoordination of second-row stereocilia length. Furthermore, Espin overexpression results in the misregulation of other stereocilia factors including GNAI3, GPSM2, EPS8, WHRN, and MYO15A, revealing that GNAI3 and GPSM2 are dispensable for stereocilia overgrowth. Finally, using an in vitro actin polymerization assay we show that espin provides an anti-capping function that requires both the G-actin binding WH2 domain as well as either the C-terminal F-actin binding domain or the internal xAB actin-binding domain. Our results provide a novel function for Espins at the barbed ends of actin filaments distinct from its previous known function of actin bundling that may account for their effects on stereocilia growth.

Whole-body analysis of TRPML3 (MCOLN3) expression using a GFP-reporter mouse model reveals widespread expression in secretory cells and endocrine glands.

TRPML3 (mucolipin 3, MCOLN3) is an endolysosomal cation channel belonging to the TRPML subfamily of transient receptor potential channels. Gain-of-function mutations in the Trpml3 gene cause deafness, circling behavior and coat color dilution in mice due to cell death of TRPML3-expressing hair cells of the inner ear or skin melanocytes, respectively. Furthermore, TRPML3 was found to play a role in the long term survival of cochlear hair cells (its absence contributing to presbycusis), in specialized giant lysosomes that neonatal (birth to weaning) enterocytes used for the uptake and digestion of maternal milk nutrients, and in the expulsion of exosome-encased bacteria such as uropathogenic E. coli, infecting bladder epithelial cells. Recently, TRPML3 was found to be expressed at high levels in alveolar macrophages and loss of TRPML3 results in a lung emphysema phenotype, confirmed in two independently engineered Trpml3 knockout lines. TRPML3 is not ubiquitously expressed like its relative TRPML1 and thus cellular expression of TRPML3 on a whole-tissue level remains, with the exceptions mentioned above, largely elusive. To overcome this problem, we generated a τGFP reporter mouse model for TRPML3 and compared expression data obtained from this model by immunofluorescence on tissue sections with immunohistochemistry using TRPML3 antibodies and in situ hybridization. We thus uncovered expression in several organs and distinct cell types. We confirmed TRPML3 expression in both neonatal and adult alveolar macrophages, in melanocytes of hair follicles and glabrous skin, in principle cells of the collecting duct of the neonatal and adult kidney, and in olfactory sensory neurons of the olfactory epithelium, including its fibres protruding to the glomeruli of the olfactory bulb. Additionally, we localized TRPML3 in several glands including parathyroid, thyroid, salivary, adrenal, and pituitary gland, testes and ovaries, suggestive of potential roles for the channel in secretion or uptake of different hormones.

Lung emphysema and impaired macrophage elastase clearance in mucolipin 3 deficient mice.

Lung emphysema and chronic bronchitis are the two most common causes of chronic obstructive pulmonary disease. Excess macrophage elastase MMP-12, which is predominantly secreted from alveolar macrophages, is known to mediate the development of lung injury and emphysema. Here, we discovered the endolysosomal cation channel mucolipin 3 (TRPML3) as a regulator of MMP-12 reuptake from broncho-alveolar fluid, driving in two independently generated Trpml3-/- mouse models enlarged lung injury, which is further exacerbated after elastase or tobacco smoke treatment. Mechanistically, using a Trpml3IRES-Cre/eR26-τGFP reporter mouse model, transcriptomics, and endolysosomal patch-clamp experiments, we show that in the lung TRPML3 is almost exclusively expressed in alveolar macrophages, where its loss leads to defects in early endosomal trafficking and endocytosis of MMP-12. Our findings suggest that TRPML3 represents a key regulator of MMP-12 clearance by alveolar macrophages and may serve as therapeutic target for emphysema and chronic obstructive pulmonary disease.

Targeting of the hair cell proteins cadherin 23, harmonin, myosin XVa, espin, and prestin in an epithelial cell model.

We have developed an advantageous epithelial cell transfection model for examining the targeting, interactions, and mutations of hair cell proteins. When expressed in LLC-PK1-CL4 epithelial cells (CL4 cells), the outer hair cell protein prestin showed faithful domain-specific targeting to the basolateral plasma membrane. We examined the consequences of mutations affecting prestin activity and assigned a targeting role to the cytoplasmic tail. The stereociliary link protein cadherin 23 (Cdh23) was targeted to the plasma membrane of CL4 cell microvilli, the topological equivalent of stereocilia. In cells coexpressing the Cdh23 cytoplasmic binding protein harmonin, a large fraction of harmonin became colocalized with Cdh23 in microvilli. Using this assay and in vitro protein binding assays, we formulated an alternative model for Cdh23-harmonin binding, in which the primary interaction is between the harmonin N-domain and a 35-residue internal peptide in the Cdh23 cytoplasmic tail. Contrary to a previous model, we found no role for the Cdh23 C-terminal PDZ (PSD-95/Dlg/ZO-1)-binding motif and observed that Cdh23 bound similar levels of harmonin with or without the exon 68 peptide. We also examined two proteins involved in stereocilium elongation. The stereociliary actin-bundling protein espin was targeted to CL4 cell microvilli and caused microvillar elongation, whereas espin with the c.2469delGTCA or c.1988delAGAG human deafness mutation showed defects in microvillar targeting and elongation. The unconventional myosin motor myosin XVa accumulated at the tips of espin-elongated microvilli, by analogy to its location in stereocilia, whereas myosin XVa with the c.4351G>A or c.4669A>G human deafness mutation did not, revealing functional deficits in motor activity.

TRPML2 and the evolution of mucolipins.

TRPML2, the polypeptide product of the gene Trpml2 (aka Mcoln2), is a member of the TRPML or mucolipin branch of the TRP super family of ion channels. Although no known agonists have been discovered, the wild type channel gives basal currents when heterologously expressed in Drosophila (S2) cells and is constitutively active in mammalian cells when bearing a cell degeneration-causing, proline to alanine substitution in the fifth trans-membrane domain. TRPML2 forms channels that are inwardly rectifying and permeable to Ca(+2), Na(+), and Fe(+2). Localization studies indicate TRPML2 is present in lysosomes, late endosomes, recycling endosomes and, at a lower level, the plasma membrane. Tissue and organ distribution of TRPML2 is solely reported through RT-PCR and it is uncertain which cell types express this channel. However, various studies suggest that lymphoid cells express TRPML2. Although the function of TRPML2 is not known, distribution and channel properties suggest it could play roles in calcium release from endolysosomes, perhaps to mediate calcium-dependent events such as vesicle fusion, or to release calcium from intracellular acidic stores. However, TRPML2 may also function in the plasma membrane and its abundance in vesicles of the endocytic pathaway might occur because its presence in the cell surface is regulated by endocytosis and exocytosis. An evolutionary analysis of Trpml2 and its relatives reveals that vertebrate and invertebrate chordates have only one Trpml gene, that Trpml1 and Trpml2 are common to vertebrates, and that Trpml3 is only found in tetrapods. Ray-finned fishes contain another isoform, which we term Trpml4 or Mcoln4 (and its product TRPML4). Trpml2 is next to Trpml3 in all tetrapod genomes except that of the frog Xenopus tropicalis and of the domesticated pig, which seems to lack most of the Trpml3 gene. This close linkage across species implies that it is maintained by selective pressure and suggests that the regulation of both genes is interdependent.

The varitint-waddler (Va) deafness mutation in TRPML3 generates constitutive, inward rectifying currents and causes cell degeneration.

Varitint-waddler (Va and Va(J)) mice are deaf and have vestibular impairment, with inner ear defects that include the degeneration and loss of sensory hair cells. The semidominant Va mutation results in an alanine-to-proline substitution at residue 419 (A419P) of the presumed ion channel TRPML3. Another allele, Va(J), has the A419P mutation in addition to an I362T mutation. We found that hair cells, marginal cells of stria vascularis, and other cells lining the cochlear and vestibular endolymphatic compartments express TRPML3. When heterologously expressed in LLC-PK1-CL4 epithelial cells, a culture model for hair cells, TRPML3 accumulated in lysosomes and in espin-enlarged microvilli that resemble stereocilia. We also demonstrated that wild-type TRPML3 forms channels that are blocked by Gd(3+), have a conductance of 50-70 pS and, like many other TRP channels, open at very positive potentials and thus rectify outwardly. In addition to this outward current, TRPML3(419P) and (I362T+A419P) generated a constitutive inwardly rectifying current that suggests a sensitivity to hyperpolarizing negative potentials and that depolarized the cells. Cells expressing TRPML3(A419P) or (I362T+A419P), but not wild-type TRPML3, died and were extruded from the epithelium in a manner reminiscent of degenerating hair cells in Va mice. The increased open probability of TRPML3(A419P) and (I362T+A419P) at physiological potentials likely underlies hair cell degeneration and deafness in Va and Va(J) mice.

Axodendritic versus axosomatic cochlear efferent termination is determined by afferent type in a hierarchical logic of circuit formation.

Hearing involves a stereotyped neural network communicating cochlea and brain. How this sensorineural circuit assembles is largely unknown. The cochlea houses two types of mechanosensory hair cells differing in function (sound transmission versus amplification) and location (inner versus outer compartments). Inner (IHCs) and outer hair cells (OHCs) are each innervated by a distinct pair of afferent and efferent neurons: IHCs are contacted by type I afferents receiving axodendritic efferent contacts; OHCs are contacted by type II afferents and axosomatically terminating efferents. Using an Insm1 mouse mutant with IHCs in the position of OHCs, we discover a hierarchical sequence of instructions in which first IHCs attract, and OHCs repel, type I afferents; second, type II afferents innervate hair cells not contacted by type I afferents; and last, afferent fiber type determines if and how efferents innervate, whether axodendritically on the afferent, axosomatically on the hair cell, or not at all.

TRPA1 is a candidate for the mechanosensitive transduction channel of vertebrate hair cells.

Mechanical deflection of the sensory hair bundles of receptor cells in the inner ear causes ion channels located at the tips of the bundle to open, thereby initiating the perception of sound. Although some protein constituents of the transduction apparatus are known, the mechanically gated transduction channels have not been identified in higher vertebrates. Here, we investigate TRP (transient receptor potential) ion channels as candidates and find one, TRPA1 (also known as ANKTM1), that meets criteria for the transduction channel. The appearance of TRPA1 messenger RNA expression in hair cell epithelia coincides developmentally with the onset of mechanosensitivity. Antibodies to TRPA1 label hair bundles, especially at their tips, and tip labelling disappears when the transduction apparatus is chemically disrupted. Inhibition of TRPA1 protein expression in zebrafish and mouse inner ears inhibits receptor cell function, as assessed with electrical recording and with accumulation of a channel-permeant fluorescent dye. TRPA1 is probably a component of the transduction channel itself.

Mutant sodium channel for tumor therapy.

Viral vectors have been used to deliver a wide range of therapeutic genes to tumors. In this study, a novel tumor therapy was achieved by the delivery of a mammalian brain sodium channel, ASIC2a, carrying a mutation that renders it constitutively open. This channel was delivered to tumor cells using a herpes simplex virus-1/Epstein-Barr virus (HSV/EBV) hybrid amplicon vector in which gene expression was controlled by a tetracycline regulatory system (tet-on) with silencer elements. Upon infection and doxycycline induction of mutant channel expression in tumor cells, the open channel led to amiloride-sensitive sodium influx as assessed by patch clamp recording and sodium imaging in culture. Within hours, tumor cells swelled and died. In addition to cells expressing the mutant channel, adjacent, noninfected cells connected by gap junctions also died. Intratumoral injection of HSV/EBV amplicon vector encoding the mutant sodium channel and systemic administration of doxycycline led to regression of subcutaneous tumors in nude mice as assessed by in vivo bioluminescence imaging. The advantage of this direct mode of tumor therapy is that all types of tumor cells become susceptible and death is rapid with no time for the tumor cells to become resistant.

A double TRPtych: six views of transient receptor potential channels in disease and health.

At the 2008 Annual Meeting of the Society for Neuroscience, a Mini-Symposium entitled "Contributions to TRP Channels to Neurological Disease" included talks from six heads of newly established laboratories, each with a unique research focus, model system, and set of experimental tools. Some of the questions addressed in these talks include the following. What is the role of transient receptor potential (TRP) channels in pain perception? How do normally functioning TRP channels contribute to cell death pathways? What are the characteristics of TRPpathies, disease states that result from overactive or underactive TRP channels? How are TRP channels regulated by signal transduction cascades? This review summarizes recent results from those laboratories and provides six perspectives on the subject of TRP channels and disease.

Auditory Neural Activity in Congenitally Deaf Mice Induced by Infrared Neural Stimulation.

To determine whether responses during infrared neural stimulation (INS) result from the direct interaction with spiral ganglion neurons (SGNs), we tested three genetically modified deaf mouse models: Atoh1-cre; Atoh1 f/f (Atoh1 conditional knockout, CKO), Atoh1-cre; Atoh1 f/kiNeurog1 (Neurog1 knockin, KI), and the Vglut3 knockout (Vglut3 -/-) mice. All animals were exposed to tone bursts and clicks up to 107 dB (re 20 µPa) and to INS, delivered with a 200 µm optical fiber. The wavelength (λ) was 1860 nm, the radiant energy (Q) 0-800 µJ/pulse, and the pulse width (PW) 100-500 µs. No auditory responses to acoustic stimuli could be evoked in any of these animals. INS could not evoke auditory brainstem responses in Atoh1 CKO mice but could in Neurog1 KI and Vglut3 -/- mice. X-ray micro-computed tomography of the cochleae showed that responses correlated with the presence of SGNs and hair cells. Results in Neurog1 KI mice do not support a mechanical stimulation through the vibration of the basilar membrane, but cannot rule out the direct activation of the inner hair cells. Results in Vglut3 -/- mice, which have no synaptic transmission between inner hair cells and SGNs, suggested that hair cells are not required.

MechanoTRPs and TRPA1.

Genetic and molecular searches in animals identify two families of ion channels used by specialized mechanosensory cells. These are the degenerin/epithelial Na+ channels (Deg/ENaCs) and transient receptor potential (TRP) channels. Some of these channels open in response to mechanical forces and/or mediate cellular responses to mechanical stimulation. TRPA1 is expressed in nociceptive neurons of peripheral ganglia and in the sensory epithelia of the inner ear. In nociceptors, TRPA1 forms chemosensitive channels that mediate the response to exogenous pain-producing chemicals as well as to the endogenous proalgesic bradykinin (BK). More indirect evidence suggests that TRPA1 might also form mechanosensory channels. Some of the TRP channels that mediate mechanical responses are not necessarily mechanically gated. For example, TRPV4 mutant mice have reduced sensitivity to noxious tactile stimulation, and heterologously expressed TRPV4 opens in response to hypotonic solution (which induces cell swelling and thus stretches membranes). TRPA1 genes in mammals are large, occupy around 50kb of chromosomal DNA and are encoded by at least 27 exons. In humans, the TRPA1 gene is located on chromosome 8q13.

Nociceptor and hair cell transducer properties of TRPA1, a channel for pain and hearing.

Mechanosensory channels of sensory cells mediate the sensations of hearing, touch, and some forms of pain. The TRPA1 (a member of the TRP family of ion channel proteins) channel is activated by pain-producing chemicals, and its inhibition impairs hair cell mechanotransduction. As shown here and previously, TRPA1 is expressed by hair cells as well as by most nociceptors (small neurons of dorsal root, trigeminal, and nodose ganglia) and localizes to their sensory terminals (mechanosensory stereocilia and peripheral free nerves, respectively). Thus, TRPA1 channels are proposed to mediate transduction in both hair cells and nociceptors. Accordingly, we find that heterologously expressed TRPA1 display channel behaviors expected for both auditory and nociceptive transducers. First, TRPA1 and the hair cell transducer share a unique set of pore properties not described for any other channel (block by gadolinium, amiloride, gentamicin, and ruthenium red, a ranging conductance of approximately 100 pS that is reduced to 54% by calcium, permeating calcium-induced potentiation followed by closure, and reopening by depolarization), supporting a direct role of TRPA1 as a pore-forming subunit of the hair cell transducer. Second, TRPA1 channels inactivate in hyperpolarized cells but remain open in depolarized cells. This property provides a mechanism for the lack of desensitization, coincidence detection, and allodynia that characterize pain by allowing a sensory neuron to respond constantly to sustained stimulation that is suprathreshold (i.e., noxious) and yet permitting the same cell to ignore sustained stimulation that is subthreshold (i.e., innocuous). Our results support a TRPA1 role in both nociceptor and hair cell transduction.