RESUMO
KEY POINTS: Vestibular type I and type II hair cells and their afferent fibres send information to the brain regarding the position and movement of the head. The characteristic feature of type I hair cells is the expression of a low-voltage-activated outward rectifying K+ current, IK,L , whose biophysical properties and molecular identity are still largely unknown. In vitro, the afferent nerve calyx surrounding type I hair cells causes unstable intercellular K+ concentrations, altering the biophysical properties of IK,L . We found that in the absence of the calyx, IK,L in type I hair cells exhibited unique biophysical activation properties, which were faithfully reproduced by an allosteric channel gating scheme. These results form the basis for a molecular and pharmacological identification of IK,L . ABSTRACT: Type I and type II hair cells are the sensory receptors of the mammalian vestibular epithelia. Type I hair cells are characterized by their basolateral membrane being enveloped in a single large afferent nerve terminal, named the calyx, and by the expression of a low-voltage-activated outward rectifying K+ current, IK,L . The biophysical properties and molecular profile of IK,L are still largely unknown. By using the patch-clamp whole-cell technique, we examined the voltage- and time-dependent properties of IK,L in type I hair cells of the mouse semicircular canal. We found that the biophysical properties of IK,L were affected by an unstable K+ equilibrium potential (Veq K+ ). Both the outward and inward K+ currents shifted Veq K+ consistent with K+ accumulation or depletion, respectively, in the extracellular space, which we attributed to a residual calyx attached to the basolateral membrane of the hair cells. We therefore optimized the hair cell dissociation protocol in order to isolate mature type I hair cells without their calyx. In these cells, the uncontaminated IK,L showed a half-activation at -79.6 mV and a steep voltage dependence (2.8 mV). IK,L also showed complex activation and deactivation kinetics, which we faithfully reproduced by an allosteric channel gating scheme where the channel is able to open from all (five) closed states. The 'early' open states substantially contribute to IK,L activation at negative voltages. This study provides the first complete description of the 'native' biophysical properties of IK,L in adult mouse vestibular type I hair cells.
Assuntos
Células Ciliadas Vestibulares/fisiologia , Ativação do Canal Iônico , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Potenciais de Ação , Regulação Alostérica , Animais , Células Cultivadas , Feminino , Células Ciliadas Vestibulares/metabolismo , Masculino , Camundongos , Canais de Potássio de Abertura Dependente da Tensão da Membrana/químicaRESUMO
Ca2+ ions play a pivotal role in inner ear hair cells as they are involved from the mechano-electrical transduction to the transmitter release. Most of the Ca2+ that enters into hair cells via mechano-transduction and voltage-gated channels is extruded by the plasma membrane Ca2+-ATPases (PMCAs) that operate in both apical and basal cellular compartments. Here, we determined the identity and distribution of PMCA isoforms in frog crista ampullaris: we showed that PMCA1, PMCA2 and PMCA3 are expressed, while PMCA4 appears to be negligible. We also identify PMCA1bx, PMCA2av and PMCA2bv as the major splice variants produced from PMCA1 and PMCA2 genes. PMCA2av appears to be the major Ca2+-pump operating at the apical pole of the cell, even if PMCA1b is also expressed in the stereocilia. PMCA1bx is, instead, the principal PMCA of hair cell basolateral compartment, where it is expressed together with PMCA2 (probably PMCA2bv) and PMCA3. Frog crista ampullaris hair cells lack a Na/Ca exchanger, therefore PMCAs are the only mechanism of Ca2+ extrusion. The coexpression of specific isozymes in the different cellular compartments responds to the need of a fine regulation of both basal and dynamic Ca2+ levels at the apical and basal pole of the cell.
Assuntos
Mecanotransdução Celular , ATPases Transportadoras de Cálcio da Membrana Plasmática/análise , Rana esculenta , Canais Semicirculares/enzimologia , Animais , Cálcio/metabolismo , Polaridade Celular , Células Epiteliais/enzimologia , Células Ciliadas Vestibulares/enzimologia , Imuno-Histoquímica , ATPases Transportadoras de Cálcio da Membrana Plasmática/genética , Isoformas de Proteínas/análise , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Several genetic mutations affecting the development and function of mammalian hair cells have been shown to cause deafness but not vestibular defects, most likely because vestibular deficits are sometimes centrally compensated. The study of hair cell physiology is thus a powerful direct approach to ascertain the functional status of the vestibular end organs. Deletion of Epidermal growth factor receptor pathway substrate 8 (Eps8), a gene involved in actin remodeling, has been shown to cause deafness in mice. While both inner and outer hair cells from Eps8 knockout (KO) mice showed abnormally short stereocilia, inner hair cells (IHCs) also failed to acquire mature-type ion channels. Despite the fact that Eps8 is also expressed in vestibular hair cells, Eps8 KO mice show no vestibular deficits. In the present study we have investigated the properties of vestibular Type I and Type II hair cells in Eps8-KO mice and compared them to those of cochlear IHCs. In the absence of Eps8, vestibular hair cells show normally long kinocilia, significantly shorter stereocilia and a normal pattern of basolateral voltage-dependent ion channels. We have also found that while vestibular hair cells from Eps8 KO mice show normal voltage responses to injected sinusoidal currents, which were used to mimic the mechanoelectrical transducer current, IHCs lose their ability to synchronize their responses to the stimulus. We conclude that the absence of Eps8 produces a weaker phenotype in vestibular hair cells compared to cochlear IHCs, since it affects the hair bundle morphology but not the basolateral membrane currents. This difference is likely to explain the absence of obvious vestibular dysfunction in Eps8 KO mice.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Células Ciliadas Auditivas Internas/metabolismo , Células Ciliadas Vestibulares/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Surdez/metabolismo , Surdez/patologia , Células Ciliadas Auditivas Internas/patologia , Células Ciliadas Vestibulares/patologia , Potenciais da Membrana/fisiologia , Camundongos Knockout , Técnicas de Patch-Clamp , Fotomicrografia , Estereocílios/metabolismo , Estereocílios/patologiaRESUMO
Hair cells, the mechanoreceptors of the acoustic and vestibular system, are presynaptic to primary afferent neurons of the eighth nerve and excite neural activity by the release of glutamate. In the present work, the role played by intracellular Ca2+ stores in afferent transmission was investigated, at the presynaptic level, by monitoring changes in the intracellular Ca2+ concentration ([Ca2+]i) in vestibular hair cells, and, at the postsynaptic level, by recording from single posterior canal afferent fibers. Application of 1-10 mm caffeine to hair cells potentiated Ca2+ responses evoked by depolarization at selected Ca2+ hot spots, and also induced a graded increase in cell membrane capacitance (DeltaCm), signaling exocytosis of the transmitter. Ca2+ signals evoked by caffeine peaked in a region located approximately 10 microm from the base of the hair cell. [Ca2+]i increases, similarly localized, were observed after 500 msec depolarizations, but not with 50 msec depolarizations, suggesting the occurrence of calcium-induced calcium release (CICR) from the same stores. Both Ca2+ and DeltaCm responses were inhibited after incubation with ryanodine (40 microm) for 8-10 min. Consistent with these results, afferent transmission was potentiated by caffeine and inhibited by ryanodine both at the level of action potentials and of miniature EPSPs (mEPSPs). Neither caffeine nor ryanodine affected the shape and amplitude of mEPSPs, indicating that both drugs acted at the presynaptic level. These results strongly suggest that endogenous modulators of the CICR process will affect afferent activity elicited by mechanical stimuli in the physiological frequency range.
Assuntos
Vias Aferentes/fisiologia , Cálcio/metabolismo , Células Ciliadas Vestibulares/metabolismo , Terminações Pré-Sinápticas/metabolismo , Animais , Cafeína/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/fisiologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/fisiologia , Capacitância Elétrica , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/fisiologia , Exocitose/efeitos dos fármacos , Corantes Fluorescentes , Células Ciliadas Vestibulares/efeitos dos fármacos , Técnicas In Vitro , Técnicas de Patch-Clamp , Rana esculenta , Rianodina/farmacologia , Transmissão Sináptica/fisiologiaRESUMO
[This corrects the article on p. 428 in vol. 8, PMID: 25565962.].
RESUMO
The present study describes the localization and distribution of putative ecto-nucleoside-triphosphate-diphosphohydrolases in the frog semicircular canals. These enzymes provide the terminating mechanism of adenosine-5'-triphosphate (ATP) signalling. The localization of the ATP hydrolysis was mapped ultracytochemically using a one-step cerium citrate reaction. Electron-dense precipitates, indicating ecto-adenosine-triphosphatase (ecto-ATPase) activity, were found at the outer surface of plasma membranes of crista hair cells and supporting cells of the sensory epithelium, transitional cells and undifferentiated cells of the ampullar wall and dark cells constituting the secretory epithelium. Non-sensory cells of the ampulla usually exhibited reaction deposits at the level of both apical and basolateral membranes coming into contact with the endolymph and the perilymph respectively, while cells constituting the sensory epithelium showed evident differences in relation to their position. Hair cells and supporting cells of the peripheral regions exhibited clear reaction products both at the level of apical and basolateral membranes, while those of the isthmus region showed abundant reactivity only at the level of their apical membranes. Of particular interest was the observation that hair cell stereocilia exhibited an abundant ecto-ATPase activity, thus suggesting a possible colocalization of enzymatic sites with purinergic receptors and mechanotransduction channels. This strategic expression of ecto-ATPase sites could provide a rapid mechanism of ATP removal able to rapidly restore the sensitivity of transduction channels. In conclusion, the widespread distribution of ecto-ATPase sites at the level of sensory and non-sensory cells of the frog semicircular canals suggests that ATP may have a key role in controlling vestibular function.
Assuntos
Adenosina Trifosfatases/metabolismo , Rana esculenta/metabolismo , Canais Semicirculares/enzimologia , Animais , Histocitoquímica , Microscopia EletrônicaRESUMO
The function of the enzyme glutamate decarboxylase (GAD) is to convert glutamate in γ-aminobutyric acid (GABA). Glutamate decarboxylase exists as two major isoforms, termed GAD65 and GAD67, that are usually expressed in GABA-containing neurons in the central nervous system. GAD65 has been proposed to be associated with GABA exocytosis whereas GAD67 with GABA metabolism. In the present immunofluorescence study, we have investigated the presence of the two GAD isoforms in the semicircular canal cristae of wild type and GAD67-GFP knock-in mice. While no evidence for GAD65 expression was found, GAD67 was detected in a distinct population of peripherally-located supporting cells, but not in hair cells or in centrally-located supporting cells. GABA, on the other hand, was found in all supporting cells. The present result indicate that only a discrete population of supporting cells use GAD67 to synthesize GABA. This is the first report of a marker that allows to distinguish two populations of supporting cells in the vestibular epithelium. On the other hand, the lack of GABA and GAD enzymes in hair cells excludes its involvement in afferent transmission.
RESUMO
The effects of acute gentamicin application on hair cells isolated from the frog semicircular canals have been tested by using the patch-clamp technique in the whole-cell configuration. Extracellular gentamicin (1 mM) mostly affected the Ca(2+) macrocurrent, I(Ca), and the Ca-dependent K(+) current, I(KCa). The drug, applied to the hair cell basolateral membrane through a fast perfusion system, produced a rapid and relevant decrease (â¼34%) of I(Ca) amplitude, without apparently affecting its activation-deactivation kinetics. The I(KCa) component of the delayed I(KD) was similarly affected: peak and steady-state mean amplitudes were significantly reduced, by about 47 and 54%, respectively, whereas the time constant of the mono-exponential current rising phase did not change. The Ca(2+) independent fraction of I(KD), I(KV), and the fast IA current were unaffected. Transduction channels (permeable to and blocked by gentamicin) are not available in the isolated hair cell, so the effect of intracellular gentamicin was tested by applying the drug through the patch pipette (1 mM in the pipette): again, it significantly reduced both I(Ca) and I(KD) amplitude, without affecting currents kinetics. IA properties were also unaffected. The drug did not affect the onset and removal of I(KD) inactivation, although the changes were scaled to the reduced I(KD) amplitude. From these observations, it is expected that hair cells exposed to gentamicin 'in vivo' become unresponsive to physiological stimulation (block of the transduction channels) and transmitter release at the cytoneural junction be drastically depressed due to reduced Ca(2+) inflow. In particular, functional impairment ensues much earlier than biochemical events that lead to hair cell apoptosis.
Assuntos
Antibacterianos/toxicidade , Canais de Cálcio/efeitos dos fármacos , Sinalização do Cálcio/efeitos dos fármacos , Gentamicinas/toxicidade , Células Ciliadas Auditivas/efeitos dos fármacos , Canais de Potássio Cálcio-Ativados/efeitos dos fármacos , Canais Semicirculares/efeitos dos fármacos , Animais , Canais de Cálcio/metabolismo , Relação Dose-Resposta a Droga , Células Ciliadas Auditivas/metabolismo , Transporte de Íons , Potenciais da Membrana , Técnicas de Patch-Clamp , Canais de Potássio Cálcio-Ativados/metabolismo , Rana esculenta , Canais Semicirculares/citologia , Canais Semicirculares/metabolismo , Fatores de TempoRESUMO
The effects of microgravity on the biophysical properties of frog labyrinthine hair cells have been examined by analyzing calcium and potassium currents in isolated cells by the patch-clamp technique. The entire, anesthetized frog was exposed to vector-free gravity in a random positioning machine (RPM) and the functional modification induced on single hair cells, dissected from the crista ampullaris, were subsequently studied in vitro. The major targets of microgravity exposure were the calcium/potassium current system and the kinetic mechanism of the fast transient potassium current, I(A). The amplitude of I(Ca) was significantly reduced in microgravity-conditioned cells. The delayed current, I(KD) (a complex of I(KV) and I(KCa)), was drastically reduced, mostly in its I(KCa) component. Microgravity also affected I(KD) kinetics by shifting the steady-state inactivation curve toward negative potentials and increasing the sensitivity of inactivation removal to voltage. As concerns the I(A), the I-V and steady-state inactivation curves were indistinguishable under normogravity or microgravity conditions; conversely, I(A) decay systematically displayed a two-exponential time course and longer time constants in microgravity, thus potentially providing a larger K(+) charge; furthermore, I(A) inactivation removal at -70 mV was slowed down. Stimulation in the RPM machine under normogravity conditions resulted in minor effects on I(KD) and, occasionally, incomplete I(A) inactivation at -40 mV. Reduced calcium influx and increased K(+) repolarizing charge, to variable extents depending on the history of membrane potential, constitute a likely cause for the failure in the afferent mEPSP discharge at the cytoneural junction observed in the intact labyrinth after microgravity conditioning.
Assuntos
Canais de Cálcio/metabolismo , Células Ciliadas Auditivas/metabolismo , Canais de Potássio/metabolismo , Rana esculenta/fisiologia , Canais Semicirculares/metabolismo , Ausência de Peso , Animais , Potenciais Pós-Sinápticos Excitadores/fisiologia , Células Ciliadas Auditivas/citologia , Potenciais da Membrana/fisiologia , Modelos Animais , Técnicas de Patch-ClampRESUMO
The complement of voltage-dependent K+ currents was investigated in hair cells of the frog crista ampullaris. The currents were recorded in transversal slices of the peripheral, intermediate and central regions of the crista by applying the patch clamp technique to cells located at different positions in the slices. Voltage-clamp recordings confirmed that cells located in each region have a distinctive complement of K+ channels. Detailed investigation of the currents in each region revealed that the complement of K+ channels in intermediate and central regions showed no variations among cells, whereas peripheral hair cells differed in the expression of two classes of A-type currents. These currents showed different kinetics of inactivation as well as steady-state inactivation properties. We termed these currents fast I(A) and slow I(A) based on their inactivation speed. The magnitude of both currents exhibited a significant gradient along the transversal axis of the peripheral regions. Fast I(A) magnitude was maximal in cells located in the external zone of the crista slice and decreased gradually to become very small in the median zone (centre) of the section, while the gradient of slow I(A) magnitude was reversed. A-type currents appear to act as a transient buffer that opposes hair cell depolarization induced by positive current injections. However, fast I(A) is partially active at the cell resting potential, while slow I(A) can be recruited only following large hyperpolarizations. Thus, two types of A currents are differentially distributed in vestibular hair cells and have different roles in shaping receptor potential.
Assuntos
Células Ciliadas Auditivas/fisiologia , Canais de Potássio/fisiologia , Potássio/metabolismo , Vestíbulo do Labirinto/fisiologia , Animais , Epitélio/fisiologia , Cinética , Potenciais da Membrana/fisiologia , Técnicas de Cultura de Órgãos , Técnicas de Patch-Clamp , Rana esculenta , Vestíbulo do Labirinto/citologiaRESUMO
The presence and functional role of inositol trisphosphate receptors (IP3R) was investigated by electrophysiology and immunohistochemistry in hair cells from the frog semicircular canal. Intracellular recordings were performed from single fibres of the posterior canal in the isolated, intact frog labyrinth, at rest and during rotation, in the presence of IP3 receptor inhibitors and drugs known to produce Ca2+ release from the internal stores or to increase IP3 production. Hair cell immunolabelling for IP3 receptor was performed by standard procedures. The drug 2-aminoethoxydiphenyl borate (2APB), an IP3 receptor inhibitor, produced a marked decrease of mEPSP and spike frequency at low concentration (0.1 mm), without affecting mEPSP size or time course. At high concentration (1 mm), 2APB is reported to block the sarcoplasmic-endoplasmic reticulum Ca2+-ATPase (SERCA pump) and increase [Ca2+]i; at the labyrinthine cytoneural junction, it greatly enhanced the resting and mechanically evoked sensory discharge frequency. The selective agonist of group I metabotropic glutamate receptors (RS)-3,5-dihydroxyphenylglycine (DHPG, 0.6 mm), produced a transient increase in resting mEPSP and spike frequency at the cytoneural junction, with no effects on mEPSP shape or amplitude. Pretreatment with cyclopiazonic acid (CPA, 0.1 mm), a SERCA pump inhibitor, prevented the facilitatory effect of both 2APB and DHPG, suggesting a link between Ca2+ release from intracellular stores and quantal emission. Consistently, diffuse immunoreactivity for IP3 receptors was observed in posterior canal hair cells. Our results indicate the presence and a possibly relevant functional role of IP3-sensitive stores in controlling [Ca2+]i and modulating the vestibular discharge.
Assuntos
Canais de Cálcio/fisiologia , Células Ciliadas Vestibulares/metabolismo , Receptores Citoplasmáticos e Nucleares/fisiologia , Potenciais de Ação , Animais , Compostos de Boro/farmacologia , Cálcio/metabolismo , Canais de Cálcio/metabolismo , ATPases Transportadoras de Cálcio/antagonistas & inibidores , Potenciais Pós-Sinápticos Excitadores , Glicina/análogos & derivados , Glicina/farmacologia , Células Ciliadas Vestibulares/efeitos dos fármacos , Imuno-Histoquímica , Indóis/farmacologia , Receptores de Inositol 1,4,5-Trifosfato , Espaço Intracelular/metabolismo , Rana esculenta , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores de Glutamato Metabotrópico/agonistas , Resorcinóis/farmacologia , Retículo Sarcoplasmático/metabolismo , Canais Semicirculares/citologia , Canais Semicirculares/efeitos dos fármacos , Canais Semicirculares/metabolismoRESUMO
Ca(2+) currents in hair cells of the frog crista ampullaris were studied using the whole-cell patch-clamp technique. Currents were recorded in situ from hair cells in peripheral, intermediate and central regions of the sensory epithelium. Two types of Ca(2+) currents were found: a partially inactivating current that was expressed by nearly all central cells and by about 65% of intermediate and peripheral cells, and a sustained current expressed by the remaining cell population. The mean Ca(2+) current amplitude was larger in intermediate cells than in central or peripheral cells. The two types of Ca(2+) currents were composed of two components: a large, nifedipine-sensitive (NS) current and a small, nifedipine-insensitive (NI) current. The latter was resistant to SNX-482, omega-conotoxin MVIIC and omega-agatoxin IVA and to omega-conotoxin GVIA, antagonists of R, P/Q and N-type Ca(2+) channels. The amplitude of NS and NI currents varied among peripheral cells, where the current density gradually increased from the beginning of the region toward its end. No significant variation of Ca(2+) current density was detected in hair cells of either intermediate or central regions. These results demonstrate the presence of regional and intraregional variations in the expression of L and non-L Ca(2+) channels in the frog crista ampullaris. Finally, immunocytochemical investigations revealed the presence of Ca(2+) channel subunits of the alpha(1D) type and the unexpected expression of alpha(1B)-subunits.