RESUMEN
Dramatic structural changes in microtubules (MT) and the assembly of complicated intercellular connections are seen during the development of the cellular matrix of the sense organ for hearing, the organ of Corti. This report examines the expression of marshalin, a minus-end binding protein, during this process of cochlear development. We discovered that marshalin is abundantly expressed in both sensory hair cells and supporting cells. In the adult, prominent marshalin expression is observed in the cuticular plates of hair cells and in the noncentrosomal MT organization centers (MTOC) of Deiters' and pillar cells. Based upon differences in marshalin expression patterns seen in the organ of Corti, we identified eight isoforms ranging from 863 to 1280 amino acids. mRNAs/proteins associated with marshalin's isoforms are detected at different times during development. These isoforms carry various protein-protein interacting domains, including coiled-coil (CC), calponin homology (CH), proline-rich (PR), and MT-binding domains, referred to as CKK. We, therefore, examined membranous organelles and structural changes in the cytoskeleton induced by expressing two of these marshalin isoforms in vitro. Long forms containing CC and PR domains induce thick, spindle-shaped bundles, whereas short isoforms lacking CC and PR induce more slender variants that develop into densely woven networks. Together, these data suggest that marshalin is closely associated with noncentrosomal MTOCs, and may be involved in MT bundle formation in supporting cells. As a scaffolding protein with multiple isoforms, marshalin is capable of modifying cytoskeletal networks, and consequently organelle positioning, through interactions with various protein partners present in different cells.
RESUMEN
Sound stimuli elicit movement of the stereocilia that make up the hair bundle of cochlear hair cells, putting tension on the tip links connecting the stereocilia and thereby opening mechanotransducer (MT) channels. Tmc1 and Tmc2, two members of the transmembrane channel-like family, are necessary for mechanotransduction. To assess their precise role, we recorded MT currents elicited by hair bundle deflections in mice with null mutations of Tmc1, Tmc2, or both. During the first postnatal week, we observed a normal MT current in hair cells lacking Tmc1 or Tmc2; however, in the absence of both isoforms, we recorded a large MT current that was phase-shifted 180°, being evoked by displacements of the hair bundle away from its tallest edge rather than toward it as in wild-type hair cells. The anomalous MT current in hair cells lacking Tmc1 and Tmc2 was blocked by FM1-43, dihydrostreptomycin, and extracellular Ca(2+) at concentrations similar to those that blocked wild type. MT channels in the double knockouts carried Ca(2+) with a lower permeability than wild-type or single mutants. The MT current in double knockouts persisted during exposure to submicromolar Ca(2+), even though this treatment destroyed the tip links. We conclude that the Tmc isoforms do not themselves constitute the MT channel but are essential for targeting and interaction with the tip link. Changes in the MT conductance and Ca(2+) permeability observed in the absence of Tmc1 mutants may stem from loss of interaction with protein partners in the transduction complex.
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Sordera/fisiopatología , Células Ciliadas Auditivas Externas/fisiología , Proteínas de la Membrana/fisiología , Órgano Espiral/fisiología , Animales , Animales Recién Nacidos , Animales no Consanguíneos , Sordera/genética , Células Ciliadas Auditivas Externas/ultraestructura , Mecanorreceptores/fisiología , Mecanorreceptores/ultraestructura , Potenciales de la Membrana/fisiología , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos CBA , Ratones Noqueados , Microscopía Electrónica de Rastreo , Modelos BiológicosRESUMEN
Mechanotransduction in the mammalian auditory system depends on mechanosensitive channels in the hair bundles that project from the apical surface of the sensory hair cells. Individual stereocilia within each bundle contain a core of tightly packed actin filaments, whose length is dynamically regulated during development and in the adult. We show that the actin-binding protein epidermal growth factor receptor pathway substrate 8 (Eps8)L2, a member of the Eps8-like protein family, is a newly identified hair bundle protein that is localized at the tips of stereocilia of both cochlear and vestibular hair cells. It has a spatiotemporal expression pattern that complements that of Eps8. In the cochlea, whereas Eps8 is essential for the initial elongation of stereocilia, Eps8L2 is required for their maintenance in adult hair cells. In the absence of both proteins, the ordered staircase structure of the hair bundle in the cochlea decays. In contrast to the early profound hearing loss associated with an absence of Eps8, Eps8L2 null-mutant mice exhibit a late-onset, progressive hearing loss that is directly linked to a gradual deterioration in hair bundle morphology. We conclude that Eps8L2 is required for the long-term maintenance of the staircase structure and mechanosensory function of auditory hair bundles. It complements the developmental role of Eps8 and is a candidate gene for progressive age-related hearing loss.
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Células Ciliadas Auditivas/patología , Pérdida Auditiva/genética , Proteínas de Microfilamentos/deficiencia , Análisis de Varianza , Animales , Audiometría de Respuesta Evocada , Células Ciliadas Auditivas/fisiología , Células Ciliadas Auditivas/ultraestructura , Ratones , Ratones Noqueados , Proteínas de Microfilamentos/genética , Microscopía Electrónica , Técnicas de Placa-ClampRESUMEN
The key components of acousticolateralis systems (lateral line, hearing and balance) are sensory hair cells. At their apex, these cells have a bundle of specialized cellular protrusions, which are modified actin-containing microvilli, connected together by extracellular filaments called cross links. Stereociliary deflections open nonselective cation channels allowing ions from the extracellular environment into the cell, a process called mechanoelectrical transduction. This produces a receptor potential that causes the release of the excitatory neurotransmitter glutamate onto the terminals of the sensory nerve fibres, which connect to the cell base, causing nerve signals to be sent to the brain. Identification of the cellular mechanisms underlying mechanoelectrical transduction and of some of the proteins involved has been assisted by research into the genetics of deafness, molecular biology and mechanical measurements of function. It is thought that one type of cross link, the tip link, is composed of cadherin 23 and protocadherin 15, and gates the transduction channel when the bundle is deflected. Another type of link, called lateral (or horizontal) links, maintains optimal bundle cohesion and stiffness for transduction. This Commentary summarizes the information currently available about the structure, function and composition of the links and how they might be relevant to human hearing impairment.
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Células Ciliadas Auditivas/metabolismo , Mecanotransducción Celular/fisiología , Animales , Cadherinas/metabolismo , Humanos , Transducción de Señal/fisiología , Vertebrados/metabolismoRESUMEN
Calcium is tightly regulated in cochlear outer hair cells (OHCs). It enters mainly via mechanotransducer (MT) channels and is extruded by the plasma membrane calcium ATPase (PMCA)2 isoform of the PMCA, mutations in which cause hearing loss. To assess how pump expression matches the demands of Ca(2+) homeostasis, the distribution of PMCA2 at different cochlear locations during development was quantified using immunofluorescence and post-embedding immunogold labeling. The PMCA2 isoform was confined to stereociliary bundles, first appearing at the base of the cochlea around post-natal day (P)0 followed by the middle and then the apex by P3, and was unchanged after P8. The developmental appearance matched the maturation of the MT channels in rat OHCs. High-resolution immunogold labeling in adult rats showed that PMCA2 was distributed along the membranes of all three rows of OHC stereocilia at similar densities and at about a quarter of the density in inner hair cell stereocilia. The difference between OHCs and inner hair cells was similar to the ratio of their MT channel resting open probabilities. Gold particle counts revealed no difference in PMCA2 density between low- and high-frequency OHC bundles despite larger MT currents in high-frequency OHCs. The PMCA2 density in OHC stereocilia was determined in low- and high-frequency regions from calibration of immunogold particle counts as 2200/µm(2) from which an extrusion rate of â¼200 ions/s per pump was inferred. The limited ability of PMCA2 to extrude the Ca(2+) load through MT channels may constitute a major cause of OHC vulnerability and high-frequency hearing loss.
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Cóclea/crecimiento & desarrollo , Células Ciliadas Auditivas Externas/química , ATPasas Transportadoras de Calcio de la Membrana Plasmática/análisis , Animales , Cóclea/química , Cóclea/citología , Células Ciliadas Auditivas Internas/química , Células Ciliadas Auditivas Internas/ultraestructura , Células Ciliadas Auditivas Externas/ultraestructura , Inmunohistoquímica , Ratas , Estereocilios/ultraestructuraRESUMEN
Hair cells of the mammalian cochlea are specialized for the dynamic coding of sound stimuli. The transduction of sound waves into electrical signals depends upon mechanosensitive hair bundles that project from the cell's apical surface. Each stereocilium within a hair bundle is composed of uniformly polarized and tightly packed actin filaments. Several stereociliary proteins have been shown to be associated with hair bundle development and function and are known to cause deafness in mice and humans when mutated. The growth of the stereociliar actin core is dynamically regulated at the actin filament barbed ends in the stereociliary tip. We show that Eps8, a protein with actin binding, bundling, and barbed-end capping activities in other systems, is a novel component of the hair bundle. Eps8 is localized predominantly at the tip of the stereocilia and is essential for their normal elongation and function. Moreover, we have found that Eps8 knockout mice are profoundly deaf and that IHCs, but not OHCs, fail to mature into fully functional sensory receptors. We propose that Eps8 directly regulates stereocilia growth in hair cells and also plays a crucial role in the physiological maturation of mammalian cochlear IHCs. Together, our results indicate that Eps8 is critical in coordinating the development and functionality of mammalian auditory hair cells.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Extensiones de la Superficie Celular/metabolismo , Cóclea/fisiología , Proteínas del Citoesqueleto/metabolismo , Células Ciliadas Auditivas/metabolismo , Estimulación Acústica , Potenciales de Acción , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Calcio/metabolismo , Canales de Calcio/metabolismo , Extensiones de la Superficie Celular/ultraestructura , Cóclea/citología , Cóclea/crecimiento & desarrollo , Proteínas del Citoesqueleto/genética , Sordera/genética , Potenciales Evocados Auditivos del Tronco Encefálico , Exocitosis , Eliminación de Gen , Células Ciliadas Auditivas/ultraestructura , Mecanotransducción Celular , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Canales de Potasio/metabolismoRESUMEN
The transition between the central (CNS) and peripheral nervous system (PNS) in cranial and spinal nerve roots, referred to here as the CNS-PNS border, is of relevance to nerve root disorders and factors that affect peripheral-central regeneration. Here, this border is described in the cat cochlear nerve using light microscopical sections, and scanning electron microscopy of the CNS-PNS interfaces exposed by fracture of the nerve either prior to or following critical point drying. The CNS-PNS border represents an abrupt change in type of myelin, supporting elements, and vascularization. Because central myelin is formed by oligodendrocytes and peripheral myelin by Schwann cells, the myelinated fibers are as a rule equipped with a node of Ranvier at the border passage. The border is shallower and smoother in cat cochlear nerve than expected from other nerves, and the borderline nodes are largely in register. The loose endoneurial connective tissue of the PNS compartment is closed at the border by a compact glial membrane, the mantle zone, of the CNS compartment. The mantle zone is penetrated by the nerve fibers, but is otherwise composed of astrocytes and their interwoven processes like the external limiting membrane of the brain surface with which it is continuous. The distal surface of the mantle zone is covered by a fenestrated basal lamina. Only occasional vessels traverse the border. From an anatomical point of view, the border might be expected to be a weak point along the cochlear nerve and thus vulnerable to trauma. In mature animals, the CNS-PNS border presents a barrier to regrowth of regenerating nerve fibers and to invasion of the CNS by Schwann cells. An understanding of this region in the cochlear nerve is therefore relevant to head injuries that lead to hearing loss, to surgery on acoustic Schwannomas, and to the possibility of cochlear nerve regeneration.
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Sistema Nervioso Central/ultraestructura , Nervio Coclear/ultraestructura , Microscopía Electrónica de Rastreo , Sistema Nervioso Periférico/ultraestructura , Animales , Astrocitos/ultraestructura , Gatos , Sistema Nervioso Central/citología , Nervio Coclear/citología , Disección , Femenino , Técnicas de Preparación Histocitológica , Masculino , Fibras Nerviosas/ultraestructura , Neuroglía/ultraestructura , Sistema Nervioso Periférico/citología , Células de Schwann/ultraestructura , Raíces Nerviosas Espinales/ultraestructuraRESUMEN
MicroRNAs (miRNAs) are small noncoding RNAs able to regulate a broad range of protein-coding genes involved in many biological processes. miR-96 is a sensory organ-specific miRNA expressed in the mammalian cochlea during development. Mutations in miR-96 cause nonsyndromic progressive hearing loss in humans and mice. The mouse mutant diminuendo has a single base change in the seed region of the Mir96 gene leading to widespread changes in the expression of many genes. We have used this mutant to explore the role of miR-96 in the maturation of the auditory organ. We found that the physiological development of mutant sensory hair cells is arrested at around the day of birth, before their biophysical differentiation into inner and outer hair cells. Moreover, maturation of the hair cell stereocilia bundle and remodelling of auditory nerve connections within the cochlea fail to occur in miR-96 mutants. We conclude that miR-96 regulates the progression of the physiological and morphological differentiation of cochlear hair cells and, as such, coordinates one of the most distinctive functional refinements of the mammalian auditory system.
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Diferenciación Celular/fisiología , Células Ciliadas Auditivas Internas/metabolismo , Células Ciliadas Auditivas Externas/metabolismo , MicroARNs/metabolismo , Animales , Células Ciliadas Auditivas Internas/ultraestructura , Células Ciliadas Auditivas Externas/ultraestructura , Ratones , Ratones Mutantes , MicroARNs/genética , Mutación , Especificidad de ÓrganosRESUMEN
Outer hair cells (OHCs) of the mammalian cochlea besides being sensory receptors also generate force to amplify sound-induced displacements of the basilar membrane thus enhancing auditory sensitivity and frequency selectivity. This force generation is attributable to the voltage-dependent contractility of the OHCs underpinned by the motile protein, prestin. Prestin is located in the basolateral wall of OHCs and is thought to alter its conformation in response to changes in membrane potential. The precise ultrastructural distribution of prestin was determined using post-embedding immunogold labelling and the density of the labelling was compared in low-frequency and high-frequency regions of the cochlea. The labelling was confined to the basolateral plasma membrane in hearing rats but declined towards the base of the cells below the nucleus. In pre-hearing animals, prestin labelling was lower in the membrane and also occurred in the cytoplasm, presumably reflecting its production during development. The densities of labelling in low-frequency and high-frequency regions of the cochlea were similar. Non-linear capacitance, thought to reflect charge movements during conformational changes in prestin, was measured in OHCs in isolated cochlear coils of hearing animals. The OHC non-linear capacitance in the same regions assayed in the immunolabelling was also similar in both the apex and base, with charge densities of 10,000/microm(2) expressed relative to the lateral membrane area. The results suggest that prestin density, and by implication force production, is similar in low-frequency and high-frequency OHCs.
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Proteínas de Transporte de Anión/metabolismo , Cóclea/metabolismo , Células Ciliadas Auditivas Externas/metabolismo , Acústica , Animales , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Cóclea/crecimiento & desarrollo , Cóclea/ultraestructura , Citoplasma/metabolismo , Citoplasma/ultraestructura , Capacidad Eléctrica , Células Ciliadas Auditivas Externas/ultraestructura , Audición/fisiología , Immunoblotting , Inmunohistoquímica , Técnicas In Vitro , Potenciales de la Membrana/fisiología , Microscopía Electrónica , Dinámicas no Lineales , Ratas , Ratas Sprague-Dawley , Transportadores de SulfatoRESUMEN
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RESUMEN
Our sense of hearing and balance relies on the very rapid gating of mechanotransducer channels known to be located close to the tops of the hair cell stereocilia within the stereociliary bundle. The molecular identity of the channels is unknown but functional aspects such as permeation, block and sensitivity to bundle displacement are well known. The channel has high calcium permeability and this feature has been used in conjunction with fast confocal calcium imaging to unambiguously localise the channels at the top of the two shorter rows of stereocilia in mammalian cochlear hair cells. The data suggest that they are completely absent from the tallest row. It is thought that the structures connecting stereocilia in adjacent rows, the tip links, are either directly responsible for the channel's mechanical gating, or are closely associated with the gating process. The channels must therefore be associated with the bottom part of the tip links and not the top. This feature has important implications for both the channel's gating mechanism and its regulatory adaptation mechanism. The tip link remains an attractive candidate for mechanical coupling between the bundle and the channel or an accessory protein. The localisation of the mechanotransducer channels to the lower end of the tip link represents an important milestone in the journey towards eventual identification of the channel and its gating mechanism.
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Células Ciliadas Auditivas/fisiología , Activación del Canal Iónico/fisiología , Canales Iónicos/fisiología , Mecanorreceptores/fisiología , Mecanotransducción Celular/fisiología , Animales , HumanosRESUMEN
The sensory bundle of vertebrate cochlear hair cells consists of actin-containing stereocilia that are thought to bend at their ankle during mechanical stimulation. Stereocilia have dense rootlets that extend through the ankle region to anchor them into the cuticular plate. Because this region may be important in bundle stiffness and durability during prolonged stimulation at high frequencies, we investigated the structure and dimensions of rootlets relative to the stereocilia in apical (low-frequency) and basal (high-frequency) regions of rodent cochleae using light and electron microscopy. Their composition was investigated using postembedding immunogold labeling of tropomyosin, spectrin, beta-actin, gamma-actin, espin, and prestin. The rootlets have a thick central core that widens at the ankle, and are embedded in a filamentous meshwork in the cuticular plate. Within a particular frequency region, rootlet length correlates with stereociliary height but between regions it changes disproportionately; apical stereocilia are, thus, approximately twice the height of basal stereocilia in equivalent rows, but rootlet lengths increase much less. Some rootlets contact the tight junctions that underlie the ends of the bundle. Rootlets contain spectrin, tropomyosin, and beta- and gamma-actin, but espin was not detected; spectrin is also evident near the apical and junctional membranes, whereas prestin is confined to the basolateral membrane below the junctions. These data suggest that rootlets strengthen the ankle region to provide durability and may contact with the lateral wall either to give additional anchoring of the stereocilia or to provide a route for interactions between the bundle and the lateral wall.
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Membrana Basilar/fisiología , Membrana Basilar/ultraestructura , Células Ciliadas Auditivas/fisiología , Células Ciliadas Auditivas/ultraestructura , Animales , Vías Auditivas/fisiología , Vías Auditivas/ultraestructura , Cilios/fisiología , Cilios/ultraestructura , Cóclea/fisiología , Cóclea/ultraestructura , Cobayas , Ratones , Ratas , Ratas Sprague-DawleyRESUMEN
Sound stimuli are detected in the cochlea by opening of hair cell mechanotransducer (MT) channels, one of the few ion channels not yet conclusively identified at a molecular level. To define their performance in situ, we measured MT channel properties in inner hair cells (IHCs) and outer hair cells (OHCs) at two locations in the rat cochlea tuned to different characteristic frequencies (CFs). The conductance (in 0.02 mM calcium) of MT channels from IHCs was estimated as 260 pS at both low-frequency and mid-frequency positions, whereas that from OHCs increased with CFs from 145 to 210 pS. The combination of MT channel conductance and tip link number, assayed from scanning electron micrographs, accounts for variation in whole-cell current amplitude for OHCs and its invariance for IHCs. Channels from apical IHCs and OHCs having a twofold difference in unitary conductance were both highly calcium selective but were distinguishable by a small but significant difference in calcium permeability and in their response to lowering ionic strength. The results imply that the MT channel has properties possessed by few known candidates, and its diversity suggests expression of multiple isoforms.
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Calcio/fisiología , Células Ciliadas Auditivas Internas/fisiología , Células Ciliadas Auditivas Externas/fisiología , Mecanorreceptores/fisiología , Estimulación Acústica/métodos , Animales , Calcio/farmacología , Cóclea/efectos de los fármacos , Cóclea/fisiología , Cóclea/ultraestructura , Células Ciliadas Auditivas Internas/efectos de los fármacos , Células Ciliadas Auditivas Internas/ultraestructura , Células Ciliadas Auditivas Externas/efectos de los fármacos , Células Ciliadas Auditivas Externas/ultraestructura , Canales de Potasio de Gran Conductancia Activados por el Calcio/fisiología , Canales de Potasio de Gran Conductancia Activados por el Calcio/ultraestructura , Mecanorreceptores/ultraestructura , Ratas , Ratas Sprague-DawleyRESUMEN
Cochlear hair cells respond with phenomenal speed and sensitivity to sound vibrations that cause submicron deflections of their hair bundle. Outer hair cells are not only detectors, but also generate force to augment auditory sensitivity and frequency selectivity. Two mechanisms of force production have been proposed: contractions of the cell body or active motion of the hair bundle. Here, we describe recently identified proteins involved in the sensory and motor functions of auditory hair cells and present evidence for each force generator. Both motor mechanisms are probably needed to provide the high sensitivity and frequency discrimination of the mammalian cochlea.
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Percepción Auditiva/fisiología , Movimiento Celular/fisiología , Células Ciliadas Auditivas/fisiología , Audición/fisiología , Animales , Vías Auditivas/fisiología , Humanos , Modelos Biológicos , Estimulación Física/métodosRESUMEN
Calcium buffers are important for shaping and localizing cytoplasmic Ca2+ transients in neurons. We measured the concentrations of the four main calcium-buffering proteins (calbindin-D28k, calretinin, parvalbumin-alpha, and parvalbumin-beta) in rat cochlear hair cells in which Ca2+ signaling is a central element of fast transduction and synaptic transmission. The proteins were quantified by calibrating immunogold tissue counts against gels containing known amounts of each protein, and the method was verified by application to Purkinje cells in which independent estimates exist for some of the protein concentrations. The results showed that, in animals with fully developed hearing, inner hair cells had 110 of the proteinaceous calcium buffer of outer hair cells in which the cell body contained parvalbumin-beta (oncomodulin) and calbindin-D28k at levels equivalent to 5 mm calcium-binding sites. Both proteins were partially excluded from the hair bundles, which may permit fast unbuffered Ca2+ regulation of the mechanotransducer channels. The sum of the calcium buffer concentrations decreased in inner hair cells and increased in outer hair cells as the cells developed their adult properties during cochlear maturation. The results suggest that Ca2+ has distinct roles in the two types of hair cell, reflecting their different functions in auditory transduction. Ca2+ is used in inner hair cells primarily for fast phase-locked synaptic transmission, whereas Ca2+ may be involved in regulating the motor capability underlying cochlear amplification of the outer hair cell. The high concentration of calcium buffer in outer hair cells, similar only to skeletal muscle, may protect against deleterious consequences of Ca2+ loading after acoustic overstimulation.
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Proteínas de Unión al Calcio/química , Proteínas de Unión al Calcio/metabolismo , Células Ciliadas Auditivas/química , Células Ciliadas Auditivas/metabolismo , Factores de Edad , Animales , Tampones (Química) , Proteínas de Unión al Calcio/ultraestructura , Cóclea/química , Cóclea/metabolismo , Cóclea/ultraestructura , Femenino , Células Ciliadas Auditivas/ultraestructura , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
Hair cells of the inner ear contain high concentrations of calcium-binding proteins that limit calcium signals and prevent cross talk between different signaling pathways during auditory transduction. Using light microscope immunofluorescence and post-embedding immunogold labeling in the electron microscope, we characterized the distribution of three calcium-buffering proteins in the turtle cochlea. Both calbindin-D28k and parvalbumin-beta were confined to hair cells in which they showed a similar distribution, whereas calretinin was present mainly in hair-cell nuclei but also occurred in supporting cells and nerve fibers. The hair-cell concentration of calbindin-D28k but not of parvalbumin-beta increased from the low- to high-frequency end of the cochlea. Calibration against standards containing known amounts of calcium-buffering protein processed in the same fluid drop as the cochlear sections gave cytoplasmic concentrations of calbindin-D28k as 0.13-0.63 mm and parvalbumin-beta as approximately 0.25 mm, but calretinin was an order of magnitude less. Total amount of Ca 2+-binding sites on the proteins is at least 1.0 mm in low-frequency hair cells and 3.0 mm in high-frequency cells. Reverse transcription-PCR showed that mRNA for all three proteins was expressed in turtle hair cells. We suggest that calbindin-D28k and parvalbumin-beta may serve as endogenous mobile calcium buffers, but the predominantly nuclear location of calretinin argues for another role in calcium signaling. The results support conclusions from electrophysiological measurements that millimolar concentrations of endogenous calcium buffers are present in turtle hair cells. Parvalbumin-beta was also found in both inner and outer hair cells of the guinea pig cochlea.
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Proteínas de Unión al Calcio/metabolismo , Cóclea/metabolismo , Tortugas/fisiología , Animales , Especificidad de Anticuerpos , Calbindina 2 , Calbindinas , Proteínas de Unión al Calcio/genética , Cilios/metabolismo , Cilios/ultraestructura , Cóclea/citología , Cóclea/ultraestructura , Técnica del Anticuerpo Fluorescente , Cobayas , Células Ciliadas Auditivas/metabolismo , Células Ciliadas Auditivas/ultraestructura , Inmunohistoquímica , Datos de Secuencia Molecular , Parvalbúminas/genética , Parvalbúminas/metabolismo , ARN Mensajero/biosíntesis , Proteína G de Unión al Calcio S100/genética , Proteína G de Unión al Calcio S100/metabolismoRESUMEN
The glutamate/aspartate transporter GLAST is known to occur in the plasma membrane of supporting cells and their glialike processes around the synaptic region of inner hair cells of the mammalian cochlea. Its function there is presumably to take up glutamate following the release of this putative amino acid neurotransmitter from the inner hair cells. In this study, we have investigated whether GLAST is also associated with the outer hair cells using postembedding immunogold labeling. This is interesting because it is uncertain whether the outer hair cells have a functional synapse at which glutamate may be released. However, earlier ultrastructural studies of the afferent synapses in outer hair cells in several mammalian species have shown features normally associated with synaptic activity. These observations are confirmed and extended here in guinea pig where these afferent synapses have presynaptic bodies, putative synaptic vesicles, and coated pits associated with them. Immunoreactivity for GLAST was found along the plasma membranes of Deiters' cells, especially around the synaptic region of the hair cell, on processes wrapped around approaching nerve fibers. Semiquantitative analysis of the distribution of immunogold labeling of Deiters' cells confirmed that it was densest in the region adjacent to the synapses. There was also more labeling in apical than in basal regions of the cochlea in three of the four animals examined, suggesting an association with the number of afferent synapses, which are more numerous in apical regions. Interestingly, labeling also occurred in other regions of the cell membrane away from the afferent terminals. This suggests that glutamate uptake is also required away from the immediate vicinity of synapses, perhaps as a consequence of glutamate dispersal resulting from the mechanical displacement of the cochlear partition during stimulation. Nonetheless, the particular association of GLAST with the synaptic region of the outer hair cell implies that the latter have active afferent synapses at which glutamate is released.
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Sistema de Transporte de Aminoácidos X-AG/metabolismo , Cóclea/inervación , Células Ciliadas Auditivas Externas/fisiología , Neuronas Aferentes/fisiología , Sinapsis/fisiología , Animales , Cobayas , Inmunohistoquímica , Sinapsis/ultraestructura , Distribución TisularRESUMEN
The functional effect of a monoclonal antibody, RA6.3, on mechanoelectrical transduction (MET) of outer hair cells (OHCs) isolated from the adult guinea-pig cochlea was investigated. This antibody was raised by an antiidiotypic approach against amiloride binding sites. RA6.3 irreversibly reduced the receptor current, independent of membrane potential. The time course of the functional block was independent of dilution (1:100, 50 and 10), beginning 1.2+/-0.5 min after the start of application and decreasing exponentially with a time constant of 0.29+/-0.18 min to 53+/-8% of the control current. The residual current was reversibly blocked by amiloride (300 microM), mainly at negative membrane potentials. Block of control current by amiloride was competitively inhibited by simultaneous application of RA6.3. These results suggest that RA6.3 binds to or in close proximity to amiloride receptor sites associated with the MET channels. Irreversibility, incompleteness, independence of membrane potential and independence of antibody dilution of the functional block can all be explained by irreversible binding of one antibody molecule to a receptor site, yielding a non-blocked state, followed by a relatively slow, reversible transition to a blocked state. It is proposed that the reversible transition might represent intramolecular binding of the second antibody combining site to the second receptor site.