Kinetic analysis of barium currents in chick cochlear hair cells.

Inward barium current (IBa) through voltage-gated calcium channels was recorded from chick cochlear hair cells using the whole-cell clamp technique. IBa was sensitive to dihydropyridines and insensitive to the peptide toxins omega-agatoxin IVa, omega-conotoxin GVIa, and omega-conotoxin MVIIC. Changing the holding potential over a -40 to -80 mV range had no effect on the time course or magnitude of IBa nor did it reveal any inactivating inward currents. The activation of IBa was modeled with Hodgkin-Huxley m2 kinetics. The time constant of activation, tau m, was 550 microseconds at -30 mV and gradually decreased to 100 microseconds at +50 mV. A Boltzmann fit to the activation curve, m infinity, yielded a half activation voltage of -15 mV and a steepness factor of 7.8 mV. Opening and closing rate constants, alpha m and beta m, were calculated from tau m and m infinity, then fit with modified exponential functions. The H-H model derived by evaluating the exponential functions for alpha m and beta m not only provided an excellent fit to the time course of IBa activation, but was predictive of the time course and magnitude of the IBa tail current. No differences in kinetics or voltage dependence of activation of IBa were found between tall and short hair cells. We conclude that both tall and short hair cells of the chick cochlea predominantly, if not exclusively, express noninactivating L-type calcium channels. These channels are therefore responsible for processes requiring voltage-dependent calcium entry through the basolateral cell membrane, such as transmitter release and activation of Ca(2+)-dependent K+ channels.     

Developmental expression of BK channels in chick cochlear hair cells

Background  

Cochlear hair cells are high-frequency sensory receptors. At the onset of hearing, hair cells acquire fast, calcium-activated potassium (BK) currents, turning immature spiking cells into functional receptors. In non-mammalian vertebrates, the number and kinetics of BK channels are varied systematically along the frequency-axis of the cochlea giving rise to an intrinsic electrical tuning mechanism. The processes that control the appearance and heterogeneity of hair cell BK currents remain unclear.     

A novel cholinergic receptor mediates inhibition of chick cochlear hair cells.

The central nervous system provides feedback regulation at several points within the peripheral auditory apparatus. One component of that feedback is inhibition of cochlear hair cells by release of acetylcholine (ACh) from efferent brainstem neurons. The mechanism of hair cell inhibition, and the character of the presumed cholinergic receptor, however, have eluded understanding. Both nicotinic and muscarinic, as well as some non-cholinergic ligands can affect the efferent action. We have made whole-cell, tight-seal recordings from short (outer) hair cells isolated from the chick's cochlea. These are the principal targets of cochlear efferents in birds. ACh hyperpolarizes short hair cells by opening a cation channel through which Ca2+ enters the cell and subsequently activates Ca(2+)-dependent K+ current (Fuchs & Murrow 1991, 1992). Both curare and atropine are effective-antagonists of cholinergic inhibition at 3 microM, whereas trimethaphan camsylate and strychnine block at 1 microM. The normally irreversible nicotinic antagonist, alpha-bungarotoxin, reversibly blocked the hair cell response, as did kappa-bungarotoxin. The half-blocking concentration for alpha-bungarotoxin was 26 nM. It is proposed that the hair cell AChR is a ligand-gated cation channel related to the nicotinic receptor of nerve and muscle.     

The afferent synapse of cochlear hair cells

Mechanosensory hair cells of the cochlea must serve as both transducers and presynaptic terminals, precisely releasing neurotransmitter to encode acoustic signals for the postsynaptic afferent neuron. Remarkably, each inner hair cell serves as the sole input for 10-30 individual afferent neurons, which requires extraordinary precision and reliability from the synaptic ribbons that marshal vesicular release onto each afferent. Recent studies of hair cell membrane capacitance and postsynaptic currents suggest that the synaptic ribbon may operate by simultaneous multi-vesicular release. This mechanism could serve to ensure the accurate timing of transmission, and further challenges our understanding of this synaptic nano-machine.     

利诺吡啶对豚鼠耳蜗外毛细胞和Deiters细胞钾电流的抑制

为探讨KCNQ家族钾通道在耳蜗外毛细胞和Deiters细胞的功能性表达,我们观察并记录了KCNQ家族钾通道阻滞剂利诺吡啶对豚鼠耳蜗单离外毛细胞(outer hair cells,OHCs)和Deiters细胞总钾电流的影响。采用酶孵育加机械分离法分离豚鼠耳蜗单个OHCs和Deiters细胞：运用膜片钳技术,在全细胞模式下记录正常细胞外液中8个外毛细胞和5个Deiters细胞的总钾电流,并观察100μmol／L和200μmol／L利诺吡啶对外毛细胞和Deiters细胞总钾电流的影响。结果观察到,在正常细胞外液中的单离外毛细胞,可记录到四乙基二乙胺敏感的外向性钾电流和静息膜电位附近激活的内向性钾电流(the K^ current activated at negative potential,IKa)两种钾电流,而在单离Deiters细胞中只记录到外向整流性钾电流。在细胞外液中,加入100μmol／L利诺吡啶后,OHCs中的四乙基二乙胺敏感的钾电流峰电流成分被抑制,稳态电流幅值减小,且电流的失活时问常数明显延长;在细胞外液中加入100μmol／L和200μmol／L利诺吡啶后,OHCs的内向性钾电流IKa被完全抑制;而细胞外液中利诺吡啶终浓度为200μmol／L时,Deiters细胞的外向整流性钾电流幅值无明显变化。由此我们推测,KCNQ家族钾通道存在于豚鼠耳蜗外毛细胞,其介导的钾电流是四乙基二乙胺敏感的钾电流的组成部分,并构成全部的IKn,其功能是介导细胞内K^ 外流和防止细胞过度去极化;KCNQ家族钾通道不存在于豚鼠耳蜗Dciters细胞。     

豚鼠耳蜗外毛细胞外向钾电流的研究

哺乳动物耳蜗具有超常的敏感性和频率分析能力 ,这依赖于感觉细胞基底膜的微机械反应。豚鼠耳蜗外毛细胞底侧膜存在电压依赖性Ｋ 通道、Ｃａ2 激活Ｋ 通道和内向钙通道等。文献报道牛蛙壶腹嵴毛细胞有瞬息Ｋ 电流 (ＩＡ) ,然而豚鼠耳蜗外毛细胞是否存在ＩＡ,迄今未见报道。来自脑干的内侧橄榄耳蜗束传出神经纤维大量分布于外毛细胞 ,调控着外毛细胞的功能 ,一般认为乙酰胆碱是耳蜗传出神经递质 ,此外三磷酸腺苷 (ＡＴＰ)对外毛细胞具有神经递质和神经调质双重作用 ,那么是否还有其他的递质发挥作用呢 ?我们应用全细胞膜片钳技术观察了豚鼠…     

The development of calcium and potassium currents during oogenesis in the starfish, Leptasterias hexactis

The development of membrane electrical properties of oocytes of the starfish Leptasterias hexactis during oogenesis was studied using voltage- and current-clamp techniques. Two voltage-dependent K currents--the fast transient and inwardly rectifying--are present early in oogenesis, before the rapid growth phase, and are maintained throughout oogenesis at the same current density and kinetics. The inward current, which is composed of a Ca current and a slower Ca-dependent inward sodium current, is also present early in oogenesis, but at very low current density. Late in oogenesis, after the oocyte has grown to full size, the inward current increases in amplitude by about fivefold, and undergoes major changes in kinetics. These changes are closely associated with the migration of the germinal vesicle to the cell periphery. The relationship of these events to electrophysiological changes during subsequent maturation and fertilization of the oocytes is discussed.     

The actions of calcium on hair bundle mechanics in mammalian cochlear hair cells

Sound stimuli excite cochlear hair cells by vibration of each hair bundle, which opens mechanotransducer (MT) channels. We have measured hair-bundle mechanics in isolated rat cochleas by stimulation with flexible glass fibers and simultaneous recording of the MT current. Both inner and outer hair-cell bundles exhibited force-displacement relationships with a nonlinearity that reflects a time-dependent reduction in stiffness. The nonlinearity was abolished, and hair-bundle stiffness increased, by maneuvers that diminished calcium influx through the MT channels: lowering extracellular calcium, blocking the MT current with dihydrostreptomycin, or depolarizing to positive potentials. To simulate the effects of Ca²⁺, we constructed a finite-element model of the outer hair cell bundle that incorporates the gating-spring hypothesis for MT channel activation. Four calcium ions were assumed to bind to the MT channel, making it harder to open, and, in addition, Ca²⁺ was posited to cause either a channel release or a decrease in the gating-spring stiffness. Both mechanisms produced Ca²⁺ effects on adaptation and bundle mechanics comparable to those measured experimentally. We suggest that fast adaptation and force generation by the hair bundle may stem from the action of Ca²⁺ on the channel complex and do not necessarily require the direct involvement of a myosin motor. The significance of these results for cochlear transduction and amplification are discussed.     

The calcium-activated potassium channels of turtle hair cells

A major factor determining the electrical resonant frequency of turtle cochlear hair cells is the time course of the Ca-activated K current (Art, J. J., and R. Fettiplace. 1987. Journal of Physiology. 385:207- 242). We have examined the notion that this time course is dictated by the K channel kinetics by recording single Ca-activated K channels in inside-out patches from isolated cells. A hair cell's resonant frequency was estimated from its known correlation with the dimensions of the hair bundle. All cells possess BK channels with a similar unit conductance of approximately 320 pS but with different mean open times of 0.25-12 ms. The time constant of relaxation of the average single- channel current at -50 mV in 4 microM Ca varied between cells from 0.4 to 13 ms and was correlated with the hair bundle height. The magnitude and voltage dependence of the time constant agree with the expected behavior of the macroscopic K(Ca) current, whose speed may thus be limited by the channel kinetics. All BK channels had similar sensitivities to Ca which produced half-maximal activation for a concentration of approximately 2 microM at +50 mV and 12 microM at -50 mV. We estimate from the voltage dependence of the whole-cell K(Ca) current that the BK channels may be fully activated at -35 mV by a rise in intracellular Ca to 50 microM. BK channels were occasionally observed to switch between slow and fast gating modes which raises the possibility that the range of kinetics of BK channels observed in different hair cells reflects a common channel protein whose kinetics are regulated by an unidentified intracellular factor. Membrane patches also contained 30 pS SK channels which were approximately 5 times more Ca-sensitive than BK channels at -50 mV. The SK channels may underlie the inhibitory synaptic potential produced in hair cells by efferent stimulation.     

Apamin-sensitive, small-conductance, calcium-activated potassium channels mediate cholinergic inhibition of chick auditory hair cells

Acetylcholine released from efferent neurons in the cochlea causes inhibition of mechanosensory hair cells due to the activation of calcium-dependent potassium channels. Hair cells are known to have large-conductance, “BK”-type potassium channels associated with the afferent synapse, but these channels have different properties than those activated by acetylcholine. Whole-cell (tight-seal) and cell-attached patch-clamp recordings were made from short (outer) hair cells isolated from the chicken basilar papilla (cochlea equivalent). The peptides apamin and charybdotoxin were used to distinguish the calcium-activated potassium channels involved in the acetylcholine response from the BK-type channels associated with the afferent synapse. Differential toxin blockade of these potassium currents provides definitive evidence that ACh activates apamin-sensitive, “SK”-type potassium channels, but does not activate carybdotoxin-sensitive BK channels. This conclusion is supported by tentative identification of small-conductance, calcium-sensitive but voltage-insensitive potassium channels in cell-attached patches. The distinction between these channel types is important for understanding the segregation of opposing afferent and efferent synaptic activity in the hair cell, both of which depend on calcium influx. These different calcium-activated potassium channels serve as sensitive indicators for functionally significant calcium influx in the hair cell. Accepted: 12 August 1999     

The organization of actin filaments in the stereocilia of cochlear hair cells

Within each tapering stereocilium of the cochlea of the alligator lizard is a bundle of actin filaments with > 3,000 filaments near the tip and only 18-29 filaments at the base where the bundle enters into the cuticular plate; there the filaments splay out as if on the surface of a cone, forming the rootlet. Decoration of the hair cells with subfragment 1 of myosin reveals that all the filaments in the stereocilia, including those that extend into the cuticular plate forming the rootlet, have unidirectional polarity, with the arrowheads pointing towards the cell center. The rest of the cuticular plate is composed of actin filaments that show random polarity, and numerous fine, 30 A filaments that connect the rootlet filaments to each other, to the cuticular plate, and to the membrane. A careful examination of the packing of the actin filaments in the stereocilia by thin sectin and by optical diffraction reveals that the filaments are packed in a paracrystalline array with the crossover points of all the actin helices in hear-perfect register. In transverse sections, the actin filaments are not hexagonally packed but, rather, are arranged in scalloped rows that present a festooned profile. We demonstrated that this profile is a product of the crossbridges by examining serial sections, sections of different thicknesses, and the same stereocilium at two different cutting angles. The filament packing is not altered by fixation in different media, removal of the limiting membrane by detergent extraction, or incubation of extracted hair cells in EGTA, EDTA, and Ca++ and ATP. From our results, we conclude that the stereocilia of the ear, unlike the brush border of intestinal epithelial cells, are not designed to shorten, nor do the filaments appear to slide past one another. In fact, the stereocilium is like a large, rigid structure designed to move as a lever.     

ATP-mediated potassium recycling in the cochlear supporting cells

Gap junction-mediated K⁺ recycling in the cochlear supporting cell has been proposed to play a critical role in hearing. However, how potassium ions enter into the supporting cells to recycle K⁺ remains undetermined. In this paper, we report that ATP can mediate K⁺ sinking to recycle K⁺ in the cochlear supporting cells. We found that micromolar or submicromolar levels of ATP could evoke a K⁺-dependent inward current in the cochlear supporting cells. At negative membrane potentials and the resting membrane potential of −80 mV, the amplitude of the ATP-evoked inward current demonstrated a linear relationship to the extracellular concentration of K⁺, increasing as the extracellular concentration of K⁺ increased. The inward current also increased as the concentration of ATP was increased. In the absence of ATP, there was no evoked inward current for extracellular K⁺ challenge in the cochlear supporting cells. The ATP-evoked inward current could be inhibited by ionotropic purinergic (P2X) receptor antagonists. Application of pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS, 50 μM) or pre-incubation with an irreversible P2X7 antagonist oxidized ATP (oATP, 0.1 mM) completely abolished the ATP-evoked inward current at the negative membrane potential. ATP also evoked an inward current at cell depolarization, which could be inhibited by intracellular Cs⁺ and eliminated by positive holding potentials. Our data indicate that ATP can activate P2X receptors to recycle K⁺ in the cochlear supporting cells at the resting membrane potential under normal physiological and pathological conditions. This ATP-mediated K⁺ recycling may play an important role in the maintenance of cochlear ionic homeostasis.     

Kanamycin-induced changes in cochlear hair cells of the guinea pig

Summary Electron-microscopical studies on the organ of Corti in kanamycin-intoxicated guinea pigs showed that progressive changes occurred in the cochlear hair cells. The changes appeared first in the basal turn of the organ of Corti, and affected primarily the outer hair-cells. During the later stages of intoxication, degeneration spread from the basal turn upwards, in the direction of the apex of the cochlea and, finally, affected all the external hair-cells.The early changes caused clumping of the chromatin in the nucleus, swelling of the nuclear membrane, damage in the mitochondria, with vesiculation of the mitochondrial cristae, and the formation of lamellated structures. The lysosomes were converted into dense bodies exhibiting various degeneration patterns. The ribosomes disappeared at an early stage from the cytoplasm.Even in cells where severe changes had occurred the hairs and cuticle seemed to be intact.During the final stages the plasma membrane, as well as the hairs and cuticle, was destroyed, and only remnants of these structures remained.Even after the disappearance of the sensory cells and the afferent nerve endings, some efferent nerve endings were still present in the organ of Corti.The action of kanamycin seems to differ slightly from that of streptomycin, since the changes caused by kanamycin in the plasma membrane are a fairly late effect, whereas the earliest changes occur apparently in the RNA protein-synthesis system and in the mitochondria of the external hair-cells.

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Zusammenfassung Elektronenmikroskopische Untersuchungen am Cortischen Organ von Meerschweinchen mit Kanamycinintoxikation ergaben eine fortschreitende Schädigung der cochlearen Haarzellen. Die Veränderungen begannen in der basalen Schneckenwindung und betrafen vorwiegend die äußeren Haarzellen. In späteren Intoxikationsstadien breitete sich die Degeneration von der Basalwindung nach aufwärts bis in die Schneckenspitze aus und wurde schließlich an allen äußeren Haarzellen gesehen.Die früheren Veränderungen manifestierten sich am Kern mit Chromatinverklumpung, Schwellen der Kernmembran, Schädigung der Mitochondrion mit vesikulären Cristae mitochondriales und Bildung von Lamellenstrukturen. Die Lysosomen waren in dichte Körper verwandelt und wiesen verschiedene Degenerationsmuster auf. Die Ribosomen verschwanden früh aus dem Cytoplasma. Sogar in schwer geschädigten Zellen erschienen Haare und Cuticula intakt.In den Endstadien waren Plasmamembran, Haare und Cuticula bis auf Reste zerstört. Einige efferente Nervenendigungen waren selbst nach dem Verschwinden der Haarzellen und der afferenten Endigungen noch anzutreffen.Die Wirkung von Kanamycin ist offenbar etwas anders als die von Streptomycin, da die Veränderungen an der Plasmamembran ziemlich spät auftreten, während sich die frühesten Schäden im System der RNA-Proteinsynthese und in den Mitochondrien der äußeren Haarzellen zu manifestieren scheinen.

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This investigation was supported by Grant No. NB 3956-03 from the US Public Health Service, the Swedish Medical Research Council, and Stiftelsen Therese and Johan Anderssons Minne. The skillful technical assistance of Miss Cathrine Lindholm, Miss Ann-Marie Lundberg, Miss Sonja Löfvenius, and Miss Gunilla Wiman is gratefully acknowledged. The kanamycin was kindly supplied by AB H. Lundbeck & Co.     

Early identification of inner pillar cells during rat cochlear development

Although the structure of the auditory organ in mature mammals, the organ of Corti, is clearly established, its development is far from being elucidated. Here, we examine its spatio-temporal development in rats from embryonic day 16 (E16) to E19 by using cytochemical and immunocytochemical methods at the light- and electron-microscope levels. We demonstrate that the organ of Corti develops from a non-proliferating cell zone that is located in the junctional region between two edges of the dorsal epithelium of the cochlear duct. We also reveal that the first cells to develop in this zone are the inner pillar cells, a particular type of non-sensory supporting cell, which arise in the base of the cochlear duct at the boundary between the two ridges at E16. Cell differentiation in this prosensory region continues according to a base-to-apex gradient; the inner hair cells appear in the greater epithelial ridge at E17 and the outer hair cells in the lesser epithelial ridge at E18. At E19, the various cell types of the organ of Corti are in place. Finally, we show that unlike the development of all the supporting cell types of the organ of Corti, the development of inner pillar cells within the prosensory domain seems not to involve Notch1 activation. These results highlight the central role that the inner pillar cells probably play in the development of the organ of Corti. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. This work received financial support from the Fonds de la Recherche Scientifique Médicale (grant no. 3.4540.06). N.T. is a PhD grant holder of the F.N.R.S.; B.M .is a Senior Research Associate of the F.N.R.S.     

Computational model of vectorial potassium transport by cochlear marginal cells and vestibular dark cells

Cochlear marginal cells and vestibular dark cells transport potassium into the inner ear endolymph, a potassium-rich fluid, the homeostasis of which is essential for hearing and balance. We have formulated an integrated mathematical model of ion transport across these epithelia that incorporates the biophysical properties of the major ion transporters and channels located in the apical and basolateral membranes of the constituent cells. The model is constructed for both open- and short-circuit situations to test the extremes of functional capacity of the epithelium and predicts the steady-state voltages, ion concentrations, and transepithelial currents as a function of various transporter and channel densities. We validate the model by establishing that the cells are capable of vectorial ion transport consistent with several experimental measurements. The model indicates that cochlear marginal cells do not make a significant direct contribution to the endocochlear potential and illustrates how changes to the activity of specific transport proteins lead to reduced K⁺ flux across the marginal and dark cell layers. In particular, we investigate the mechanisms of loop diuretic ototoxicity and diseases with hearing loss in which K⁺ and Cl^– transport are compromised, such as Jervell and Lange-Nielsen syndrome and Bartter syndrome, type IV, respectively. Such simulations demonstrate the utility of compartmental modeling in investigating the role of ion homeostasis in inner ear physiology and pathology. stria vascularis; endolymph; endocochlear potential; biological modeling     

Epistatic genetic interactions between Insm1 and Ikzf2 during cochlear outer hair cell development

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Potassium currents in presynaptic hair cells of Hermissenda.

Apart from their primary function as balance sensors, Hermissenda hair cells are presynaptic neurons involved in the Ca(2+)-dependent neuronal plasticity in postsynaptic B photoreceptors that accompanies classical conditioning. With a view to beginning to understand presynaptic mechanisms of plasticity in the vestibulo-visual system, a locus for conditioning-induced neuronal plasticity, outward currents that may govern the excitability of hair cells were recorded by means of a whole-cell patch-clamp technique. Three K+ currents were characterized: a 4-aminopyridine-sensitive transient outward K+ current (IA), a tetraethyl ammonium-sensitive delayed rectifier K+ current (IK,V), and a Ca(2+)-activated K+ current (IK,Ca). IA activates and decays rapidly; the steady-state activation and inactivation curves of the current reveal a window current close to the apparent resting voltage of the hair cells, suggesting that the current is partially activated at rest. By modulating firing frequency and perhaps damping membrane oscillations, IA may regulate synaptic release at baseline. In contrast, IK,V and IK,Ca have slow onset and exhibit little or no inactivation. These two K+ currents may determine the duration of the repolarization phase of hair-cell action potentials and hence synaptic release via Ca2+ influx through voltage-gated Ca2+ channels. In addition, IK,Ca may be responsible for the afterhyperpolarization of hair cell membrane voltage following prolonged stimulation.     

Elevated potassium shortens action potential duration by altering outward currents in chick dorsal root ganglia neurons

Dissociated embryonic chick dorsal root ganglion (DRG) neurons maintained in culture exhibit a mixed Na+/Ca2+ action potential. The characteristic "shoulder" on the repolarizing phase is due to the relatively prolonged inward Ca2+ current. DRG neurons grown in an elevated K+ medium (25 versus. 5 mM) lack the plateau phase of the action potential. Voltage-clamp analysis showed that this plastic change in action potential duration is not due to the loss of the inward Ca2+ current but is partly due to the appearance of a Ca2(+)-dependent, 4-aminopyridine-(4-AP)-sensitive transient outward current. Faster activation of the purely voltage-dependent delayed rectifier outward current also contributes to the rapid repolarization observed in neurons cultured in elevated K+ medium.