RESUMO
The motor protein, prestin, situated in the basolateral plasma membrane of cochlear outer hair cells (OHCs), underlies the generation of somatic, voltage-driven mechanical force, the basis for the exquisite sensitivity, frequency selectivity and dynamic range of mammalian hearing. The molecular and structural basis of the ontogenetic development of this electromechanical force has remained elusive. The present study demonstrates that this force is significantly reduced when the immature subcellular distribution of prestin found along the entire plasma membrane persists into maturity, as has been described in previous studies under hypothyroidism. This observation suggests that cochlear amplification is critically dependent on the surface expression and distribution of prestin. Searching for proteins involved in organizing the subcellular localization of prestin to the basolateral plasma membrane, we identified cochlear expression of a novel truncated prestin splice isoform named prestin 9b (Slc26A5d) that contains a putative PDZ domain-binding motif. Using prestin 9b as the bait in a yeast two-hybrid assay, we identified a calcium/calmodulin-dependent serine protein kinase (CASK) as an interaction partner of prestin. Co-immunoprecipitation assays showed that CASK and prestin 9b can interact with full-length prestin. CASK was co-localized with prestin in a membrane domain where prestin-expressing OHC membrane abuts prestin-free OHC membrane, but was absent from this area for thyroid hormone deficiency. These findings suggest that CASK and the truncated prestin splice isoform contribute to confinement of prestin to the basolateral region of the plasma membrane. By means of such an interaction, the basal junction region between the OHC and its Deiter's cell may contribute to efficient generation of somatic electromechanical force.
Assuntos
Proteínas de Transporte de Ânions/metabolismo , Eletricidade , Guanilato Quinases/metabolismo , Células Ciliadas Auditivas Externas/fisiologia , Fenômenos Mecânicos , Núcleo Vestibular Lateral/citologia , Núcleo Vestibular Lateral/metabolismo , Animais , Proteínas de Transporte de Ânions/análise , Proteínas de Transporte de Ânions/genética , Células Cultivadas , Feminino , Guanilato Quinases/análise , Guanilato Quinases/genética , Células HEK293 , Células Ciliadas Auditivas Externas/química , Células Ciliadas Auditivas Externas/citologia , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos , Proteínas Motores Moleculares/análise , Proteínas Motores Moleculares/genética , Proteínas Motores Moleculares/metabolismo , Ratos , Ratos Wistar , Transportadores de Sulfato , Núcleo Vestibular Lateral/químicaRESUMO
Thyroid hormone (TH) is essential for the development of hearing. Lack of TH in a critical developmental period from embryonic day 17 to postnatal day 12 (P12) in rats and mice leads to morphological and functional deficits in the organ of Corti and the auditory pathway. We investigated the effects of TH on inner hair cells (IHCs) using patch-clamp recordings, capacitance measurements, and immunocytochemistry in hypothyroid rats and athyroid Pax8-/- mice. Spontaneous and evoked Ca2+ action potentials (APs) were present in control IHCs from P3-P11 rats and vanished in parallel with the expression of a rapidly activating Ca2+- and voltage-activated K+ (BK) conductance. IHCs of hypothyroid rats and athyroid Pax8-/- mice displayed APs until the end of the third postnatal week because of threefold elevated Ca2+ currents and missing expression of BK currents. After the fourth postnatal week, some IHCs showed BK currents whereas adjacent IHCs did not, demonstrated by electrophysiology and immunocytochemistry. To test whether the prolonged spiking activity during TH deficiency may be transmitted at IHC synapses, capacitance measurements were performed in parallel to analysis of otoferlin expression, a protein thought to play an essential role in exocytosis of IHCs. Strikingly, otoferlin was absent from IHCs of hypothyroid rats but not of Pax8-/- mice, although both cell types showed exocytosis with an efficiency typical for immature IHCs. These results demonstrate for the first time a TH-dependent control of IHC spiking activity before the onset of hearing attributable to effects of TH on Ca2+ and BK channels. Moreover, they question an indispensable role of otoferlin for exocytosis in IHCs.
Assuntos
Potenciais de Ação/fisiologia , Canais de Cálcio/biossíntese , Células Ciliadas Auditivas Internas/metabolismo , Hipotireoidismo/metabolismo , Canais de Potássio/biossíntese , Animais , Animais Recém-Nascidos , Canais de Cálcio/genética , Feminino , Regulação da Expressão Gênica/fisiologia , Células Ciliadas Auditivas Internas/crescimento & desenvolvimento , Hipotireoidismo/genética , Camundongos , Camundongos Transgênicos , Canais de Potássio/genética , Gravidez , Ratos , Ratos WistarRESUMO
Outer hair cells (OHCs) are innervated by type II afferent fibers of as yet unknown function. It is still a matter of debate whether OHCs perform exocytosis. If so, they would require presynaptic Ca2+ channels at their basal poles where the type II fibers make contacts. Here we show that L-type Ca2+ channel currents (charge carrier, 10 mM Ba2+) present in neonatal OHCs [postnatal day 1 (P1) to P7] decreased from approximately 170 to approximately 50 pA at approximately the onset of hearing. Ba2+ currents could hardly be measured in mature mouse OHCs because of their high fragility, whereas in the rat, the average Ba2+ current amplitude of apical OHCs was 58 +/- 9 pA (n = 20, P19-P30) compared with that of the inner hair cells (IHCs) of 181 +/- 50 pA (n = 24, P17-P30). Properties of Ba2+ currents of mature OHCs resembled those of neonatal OHCs. One exception was the voltage dependence of activation that shifted between birth and P12 by +9 mV toward positive voltages in OHCs, whereas it remained constant in the IHCs. Ca(v)1.3-specific mRNA was detected in mature OHCs using cell-specific reverse transcription (RT)-PCR and in situ hybridization. Ca(v)1.3 protein was stained exclusively at the base of mature OHCs, in colocalization with the ribbon synapse protein CtBP2 (C-terminal binding protein 2)/RIBEYE. When current sizes were normalized to the estimated number of afferent fibers or presynaptic ribbons, comparable values for IHCs and OHCs were obtained, a finding that together with the colocalization of Ca(v)1.3 and CtBP2/RIBEYE protein strongly suggests a role for Ca(v)1.3 channels in exocytosis of mature OHCs.
Assuntos
Canais de Cálcio Tipo L/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Células Ciliadas Auditivas Externas/metabolismo , Transdução de Sinais/fisiologia , Éster Metílico do Ácido 3-Piridinacarboxílico, 1,4-Di-Hidro-2,6-Dimetil-5-Nitro-4-(2-(Trifluormetil)fenil)/farmacologia , Fatores Etários , Análise de Variância , Animais , Animais Recém-Nascidos , Bário/farmacologia , Agonistas dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo L/genética , Cloretos/metabolismo , Relação Dose-Resposta à Radiação , Estimulação Elétrica/métodos , Células Ciliadas Auditivas Internas/efeitos dos fármacos , Células Ciliadas Auditivas Internas/fisiologia , Células Ciliadas Auditivas Internas/efeitos da radiação , Células Ciliadas Auditivas Externas/crescimento & desenvolvimento , Hibridização In Situ/métodos , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Camundongos , Técnicas de Patch-Clamp/métodos , RatosRESUMO
The 10-member SLC26 gene family encodes anion exchangers of which SLC26A5 appears to be restricted to the outer hair cells of the inner ear. Here, the so-called prestin protein acts as a molecular motor, thought to be responsible for active mechanical amplification in the mammalian cochlea. We introduce special characteristics of SLC26A5 which may have relevance for other members of the family as well. As such, data point to a characteristic transcriptional control mechanism of which thyroid hormone surprisingly takes a role not only as an enhancer of expression, but also as a regulator of the subcellular redistribution of the prestin protein. Of significance for other members of the SLC26 family may be the observation that the failure of the subcellular redistribution of prestin protein prior to the onset of hearing leads to severe deficit of mature prestin function. Data will furthermore be argued in the context that prestin-related SLC26 proteins in the auditory organs of non-mammalian vertebrates and insects are widespread, possibly ancestral constituents of auditory organs and are likely to serve salient roles in mammals and across taxa.
Assuntos
Proteínas de Transporte de Ânions/metabolismo , Insetos/metabolismo , Homologia de Sequência , Vertebrados/metabolismo , Animais , Cóclea/citologia , Éxons/genética , Regulação da Expressão Gênica , Camundongos , Transporte Proteico , Proteínas/genética , Proteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Elementos de Resposta/genética , Transportadores de Sulfato , Transcrição Gênica , Peixe-ZebraRESUMO
Cochlear outer hair cells (OHCs) terminally differentiate prior to the onset of hearing. During this time period, thyroid hormone (TH) dramatically influences inner ear development. It has been shown recently that TH enhances the expression of the motor protein prestin via liganded TH receptor beta (TRbeta) while in contrast the expression of the potassium channel KCNQ4 is repressed by unliganded TRalpha1. These different mechanisms of TH regulation by TRalpha1 or TRbeta prompted us to analyse other ion channels that are required for the final differentiation of OHCs. We analysed the onset of expression of the Ca(2+) channel Ca(V)1.3, and the K(+) channels SK2 and BK and correlated the results with the regulation via TRalpha1 or TRbeta. The data support the hypothesis that proteins expressed in rodents prior to or briefly after birth like Ca(V)1.3 and prestin are either independent of TH (e.g. Ca(V)1.3) or enhanced through TRbeta (e.g. prestin). In contrast, proteins expressed in rodents later than P6 like KCNQ4 ( approximately P6), SK2 ( approximately P9) and BK ( approximately P11) are repressed through TRalpha1. We hypothesise that the precise regulation of expression of the latter genes requires a critical local TH level to overcome the TRalpha1 repression.
Assuntos
Diferenciação Celular/fisiologia , Células Ciliadas Auditivas Externas/metabolismo , Células Ciliadas Auditivas Externas/fisiologia , Canais Iônicos/biossíntese , Receptores alfa dos Hormônios Tireóideos/fisiologia , Animais , Antitireóideos , Cóclea/citologia , Cóclea/crescimento & desenvolvimento , Células Ciliadas Auditivas Internas/metabolismo , Células Ciliadas Auditivas Internas/fisiologia , Células Ciliadas Auditivas Externas/efeitos dos fármacos , Hipotireoidismo/induzido quimicamente , Hipotireoidismo/metabolismo , Imuno-Histoquímica , Metimazol , Camundongos , Camundongos Knockout , Modelos Estatísticos , Ratos , Especificidade da Espécie , Receptores alfa dos Hormônios Tireóideos/genética , Receptores beta dos Hormônios Tireóideos/genética , Receptores beta dos Hormônios Tireóideos/fisiologia , Hormônios Tireóideos/sangue , Hormônios Tireóideos/farmacologia , Regulação para Cima/fisiologia , Vestíbulo do Labirinto/metabolismoRESUMO
Mutations of the human otoferlin gene lead to an autosomal recessive nonsyndromic form of prelingual, sensorineural deafness (deafness autosomal recessive 9, DFNB9). Several studies have demonstrated expression of otoferlin in the inner ear and brain, and suggested a role of otoferlin in Ca(2+)-triggered exocytosis. So far, otoferlin expression profiles were solely based on the detection of mRNA. Here, we analysed the expression of otoferlin protein and mRNA using immunohistochemistry, in situ hybridization and RT-PCR in neonatal and mature Wistar rat tissue. In agreement with previous studies, otoferlin expression was found in the brain and in inner and vestibular hair cells. Otoferlin mRNA and protein was, however, also detected in mature outer hair cells of low-frequency processing cochlear turns and in auditory nerve fibres. In outer, inner and vestibular hair cells, otoferlin was subcellularly localized at a considerable distance from the presumed active release sites. Double-staining with the synaptic ribbon marker, C-terminal binding protein 2 (CtBP2), or the presynaptic Ca(2+)-channel, Ca(v)1.3, both assumed to mark the sites of vesicle fusion and transmitter release, did not colocalize with otoferlin expression and thus do not necessarily support a selected role of otoferlin in Ca(2+)-triggered exocytosis. The widespread distribution of otoferlin in neurons, nerve fibres and hair cells, and its subcellular distribution extending beyond the regions of synaptic vesicle fusion, i.e. coenrichment with the cytosolic Golgi matrix protein 130 (GM130) in inner hair cells or the early endosomal autoantigen 1 (EEA1) in outer hair cells support instead the idea of a more ubiquitous role of otoferlin in early/recycling endosome trans-Golgi network dynamics.
Assuntos
Encéfalo/metabolismo , Cóclea/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas de Membrana/metabolismo , Vestíbulo do Labirinto/metabolismo , Fatores Etários , Animais , Animais Recém-Nascidos , Autoantígenos/metabolismo , Western Blotting/métodos , Encéfalo/crescimento & desenvolvimento , Canais de Cálcio Tipo L/metabolismo , Cóclea/crescimento & desenvolvimento , Proteínas do Olho/metabolismo , Imunofluorescência/métodos , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Vestíbulo do Labirinto/crescimento & desenvolvimentoRESUMO
Thyroid hormone (TH or T3) and TH-receptor beta (TRbeta) have been reported to be relevant for cochlear development and hearing function. Mutations in the TRbeta gene result in deafness associated with resistance to TH syndrome. The effect of TRalpha1 on neither hearing function nor cochlear T3 target genes has been described to date. It is also uncertain whether TRalpha1 and TRbeta can act simultaneously on different target genes within a single cell. We focused on two concomitantly expressed outer hair cell genes, the potassium channel Kcnq4 and the motor protein prestin Slc26a5. In outer hair cells, TH enhanced the expression of the prestin gene through TRbeta. Simultaneously Kcnq4 expression was activated in the same cells by derepression of TRalpha1 aporeceptors mediated by an identified THresponse element, which modulates KCNQ4 promoter activity. We show that T3 target genes can differ in their sensitivity to TH receptors having the ligand either bound (holoreceptors) or not bound (aporeceptors) within single cells, and suggest a role for TRalpha1 in final cell differentiation.
Assuntos
Diferenciação Celular , Regulação da Expressão Gênica , Células Ciliadas Auditivas Externas/citologia , Canais de Potássio KCNQ/genética , Proteínas/genética , Receptores alfa dos Hormônios Tireóideos/metabolismo , Receptores beta dos Hormônios Tireóideos/metabolismo , Animais , Proteínas de Transporte de Ânions , Sequência de Bases , Células Cultivadas , Genes Dominantes/genética , Células Ciliadas Auditivas Externas/metabolismo , Humanos , Hipotireoidismo/metabolismo , Camundongos , Dados de Sequência Molecular , Mutação/genética , Regiões Promotoras Genéticas/genética , Ratos , Ratos Wistar , Elementos de Resposta/genética , Transportadores de Sulfato , Receptores alfa dos Hormônios Tireóideos/genética , Receptores beta dos Hormônios Tireóideos/genética , Hormônios Tireóideos/deficiênciaRESUMO
Ciliary neurotrophic factor (CNTF) signals via a tripartite receptor complex consisting of the glycosyl-phosphatidylinositol (GPI)-anchored CNTF receptor (CNTF-R), the leukaemia inhibitory factor receptor (LIF-R) and the interleukin-6 (IL-6) signal transducer gp130. We have recently reported that gp130 is endogenously expressed in the polarised epithelial model cell line Madin-Darby canine kidney (MDCK) and we have demonstrated a preferential basolateral localisation of this protein. In the present study we show that MDCK cells also express the LIF-R and respond to stimulation with human LIF by activation of tyrosine phosphorylation of signal transducer and activator of transcription-3 (STAT3), both however in an unpolarised fashion. This suggests that MDCK cells may be target cells for LIF. We have furthermore stably expressed the human CNTF-R in MDCK cells and by two different assays we found an apical localisation. Consistent with these findings, stimulation of CNTF-R-positive cells resulted only in an activation of STAT3 when CNTF was added apically. These data demonstrate that each subunit of the CNTF receptor complex has a distinct distribution in polarised cells which may reflect the different roles the respective cytokines play in vivo. Since it is currently believed that lipid rafts are involved in signal transduction as well as protein sorting we studied the association of the three receptor complex components with membrane rafts using different protocols. Whereas the CNTF-R cofractionated quantitatively with lipid rafts independently of the method used, gp130 and the LIF-R were found to associate with lipid rafts only partially when detergents were used for isolation. These findings could indicate that either the three receptor complex subunits are localised to the same kind of raft but with different affinities to the liquid-ordered environment, or that they are localised to different types of rafts. CNTF-, LIF-, and IL-6-dependent STAT3 activation was sensitive to the cholesterol-depleting drug methyl-beta-cyclodextrin (MCD) suggesting that the integrity of lipid rafts is important for IL-6-type cytokine-induced STAT activation.
Assuntos
Microdomínios da Membrana/química , Animais , Biotinilação , Western Blotting , Proteínas do Capsídeo , Caveolina 1 , Caveolinas/metabolismo , Linhagem Celular , Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Colesterol/metabolismo , Fator Neurotrófico Ciliar/metabolismo , Contactinas , Citocinas/metabolismo , DNA Complementar/metabolismo , Proteínas de Ligação a DNA/metabolismo , Detergentes/farmacologia , Cães , Vetores Genéticos/metabolismo , Glicosilfosfatidilinositóis/química , Células HeLa , Humanos , Imunoprecipitação , Interleucina-6/metabolismo , Lipídeos/química , Microscopia de Fluorescência , Moléculas de Adesão de Célula Nervosa/metabolismo , Receptor do Fator Neutrófico Ciliar/metabolismo , Fator de Transcrição STAT3 , Transdução de Sinais , Frações Subcelulares/metabolismo , Fatores de Tempo , Transativadores/metabolismo , Transfecção , beta-Ciclodextrinas/metabolismoRESUMO
The large conductance voltage- and Ca2+-activated potassium (BK) channel has been suggested to play an important role in the signal transduction process of cochlear inner hair cells. BK channels have been shown to be composed of the pore-forming alpha-subunit coexpressed with the auxiliary beta1-subunit. Analyzing the hearing function and cochlear phenotype of BK channel alpha-(BKalpha-/-) and beta1-subunit (BKbeta1-/-) knockout mice, we demonstrate normal hearing function and cochlear structure of BKbeta1-/- mice. During the first 4 postnatal weeks also, BKalpha-/- mice most surprisingly did not show any obvious hearing deficits. High-frequency hearing loss developed in BKalpha-/- mice only from approximately 8 weeks postnatally onward and was accompanied by a lack of distortion product otoacoustic emissions, suggesting outer hair cell (OHC) dysfunction. Hearing loss was linked to a loss of the KCNQ4 potassium channel in membranes of OHCs in the basal and midbasal cochlear turn, preceding hair cell degeneration and leading to a similar phenotype as elicited by pharmacologic blockade of KCNQ4 channels. Although the actual link between BK gene deletion, loss of KCNQ4 in OHCs, and OHC degeneration requires further investigation, data already suggest human BK-coding slo1 gene mutation as a susceptibility factor for progressive deafness, similar to KCNQ4 potassium channel mutations.