F-actin cleavage in apoptotic outer hair cells in chinchilla cochleas exposed to intense noise

Sensory transduction in the cochlea and the vestibular labyrinth depends on the cycling of K+. In the cochlea, endolymphatic K+ flows into the sensory hair cells via the apical transduction channel and is released from the hair cells into perilymph via basolateral K+ channels including KCNQ4. K+ may be taken up by fibrocytes in the spiral ligament and transported from cell to cell via gap junctions into strial intermediate cells. Gap junctions may include GJB2, GJB3 and GJB6. K+ is released from the intermediate cells into the intrastrial space via the KCNJ10 K+ channel that generates the endocochlear potential. From the intrastrial space, K+ is taken up across the basolateral membrane of strial marginal cells via the Na+/2Cl-/K+ cotransporter SLC12A2 and the Na+/K+-ATPase ATP1A1/ATP1B2. Strial marginal cells secrete K+ across the apical membrane into endolymph via the K+ channel KCNQ1/KCNE1, which concludes the cochlear cycle. A similar K+ cycle exists in the vestibular labyrinth. Endolymphatic K+ flows into the sensory hair cells via the apical transduction channel and is released from the hair cells via basolateral K+ channels including KCNQ4. Fibrocytes connected by gap junctions including GJB2 may be involved in delivering K+ to vestibular dark cells. Extracellular K+ is taken up into vestibular dark cells via SLC12A2 and ATP1A1/ATP1B2 and released into endolymph via KCNQ1/KCNE1, which concludes the vestibular cycle. The importance of K+ cycling is underscored by the fact that mutations of KCNQ1, KCNE1, KCNQ4, GJB2, GJB3 and GJB6 lead to deafness in humans and that null mutations of KCNQ1, KCNE1, KCNJ10 and SLC12A2 lead to deafness in mouse models.     

KCNQ1/KCNE1 K+ channel and P2Y4 receptor are co-expressed from the time of birth in the apical membrane of rat strial marginal cells

CONCLUSION: KCNQ1/KCNE1 K(+) channels and P2Y(4) receptors are expressed in the apical membrane of rat strial marginal cells from postnatal day 1 (P1) and maintained throughout development. OBJECTIVES: The purpose of the present study was to investigate the developmental expression of KCNQ1/KCNE1 K(+) channel and of P2Y(4), which is an important metabotropic regulator of KCNQ1/KCNE1 K(+) channel in strial marginal cells. MATERIALS AND METHODS: Sprague-Dawley rats at different stages of development (P1, P3, P5, P7, P14, and P21) were studied. The spiral ligament with the stria vascularis was detached from the cartilaginous or bony cochlea and prepared for a voltage-sensitive vibrating probe and immunohistochemistry. RESULTS: Chromanol 293B, a blocker of KCNQ1/KCNE1 K(+) channel, inhibited short-circuit currents (I ( sc )) from P1 to P21. Similarly, I ( sc ) were found to be decreased by uridine 5'-triphosphate at all ages. The antagonist profiles indicated that the apical P2Y receptor is P2Y(4) subtype. KCNQ1, KCNE1, and P2Y(4) were immunolocalized in the apical region of stria vascularis at P1.     

K(+) cycling and its regulation in the cochlea and the vestibular labyrinth

Potassium (K(+)) plays a very important role in the cochlea. K(+) is the major cation in endolymph and the charge carrier for sensory transduction and the generation of the endocochlear potential. The importance of K(+) handling in the cochlea is marked by the discovery of several forms of hereditary deafness that are due to mutations of K(+) channels. Deafness results from mutations of KCNQ4, a K(+) channel in the sensory hair cells, as well as from mutations of the gap junction proteins GJB2, GJB3 and GJB6 that may facilitate cell-to-cell movements of K(+). Deafness results also from mutations of KCNQ1 or KCNE1, subunits of a K(+) channel that carries K(+) from strial marginal cells and vestibular dark cells into endolymph. Further, deafness results from mutations of KCNJ10, a K(+) channel that generates the endocochlear potential in conjunction with the high K(+) concentration in strial intermediate cells and the low K(+) concentration in the intrastrial fluid spaces. This review details recent advances in the understanding of K(+) transport and its regulation in the cochlea and the vestibular labyrinth.     

Inner ear abnormalities in a Kcnq1 (Kvlqt1) knockout mouse: a model of Jervell and Lange-Nielsen syndrome.

HYPOTHESIS: Mice lacking functional KCNQ1 (previously known as KvLQT1) channels exhibit functional and structural abnormalities that indicate disturbed production of endolymph. BACKGROUND: Congenital deafness associated with cardiac conduction abnormalities (Jervell and Lange-Nielsen syndrome) is associated with dysfunctional KCNQ1/KCNE1 channel complex. This potassium channel plays a critical role in the production and homeostasis of endolymph by the stria vascularis. A preliminary report documented severe abnormalities of the scala media and vestibular compartments of a single mouse lacking functional KCNQ1 alleles. METHODS: Hearing thresholds were measured in three Kcnq1 knockout mice, two heterozygous mice, and one wild-type mouse by auditory brainstem response recordings using clicks, after which the temporal bones were removed. After fixation and dehydration, the ears were embedded in araldite, sectioned at 20-microm thickness, stained with toluidine blue on glass slides, and examined with the light microscope. RESULTS: Kcnq1 knockout mice were deaf and demonstrated circling behavior. They exhibited a marked atrophy of the stria vascularis, contraction of the endolymphatic compartments, and collapse and adhesion of surrounding membranes. There was a complete degeneration of the organ of Corti and an associated degeneration of the spiral ganglion. CONCLUSION: Kcnq1 knockout mice exhibit histopathologic findings that are comparable to those reported in human temporal bone cases of Jervell and Lange-Nielsen syndrome, and provide further evidence that KCNQ1 channel dysfunction can lead to congenital deafness in this syndrome.     

Time course of inner ear degeneration and deafness in mice lacking the Kir4.1 potassium channel subunit

The Kir4.1 gene (KCNJ10) encodes an inwardly rectifying K(+) channel subunit abundantly expressed in the CNS. Its expression in the mammalian inner ear has been suggested but its function in vivo in the inner ear is unknown. Because diverse human hereditary deafness syndromes are associated with mutations in K(+) channels, we examined auditory function and inner ear structure in mice with a genetically inactivated Kir4.1 K(+) channel subunit. Startle response experiments suggest that Kir4.1-/- mice are profoundly deaf, whereas Kir4.1+/- mice react like wild-type mice to acoustic stimuli. In Kir4.1-/- mice, the Reissner membrane is collapsed, the tectorial membrane is swollen, and type I hair cells and spiral ganglion neurons as well as their central processes degenerate over the first postnatal weeks. In the vestibular ganglia, neuronal cell death with apoptotic features is also observed. Immunostaining reveals that Kir4.1 is strongly expressed in stria vascularis of wild-type but not Kir4.1-/- mice. Within the spiral ganglion, Kir4.1 labeling was detected on satellite cells surrounding spiral ganglion neurons and axons. We conclude that Kir4.1 is crucial for normal development of the cochlea and hearing, via two distinct aspects of extracellular K(+) homeostasis: (1). in stria vascularis, Kir4.1 helps to generate the cochlear endolymph; and (2). in spiral and vestibular ganglia, Kir4.1 in surrounding glial cells helps to support the spiral and vestibular ganglion neurons and their projecting axons.     

Ca(2+)-activated nonselective cation, maxi K+ and Cl- channels in apical membrane of marginal cells of stria vascularis.

Patch clamp recordings on the apical membrane of marginal cells of the stria vascularis of the gerbil were made in the cell-attached and excised configuration. Marginal cells are thought to secrete K+ into and absorb Na+ from endolymph. Four types of channel were identified; the most frequently observed channel was a small, nonselective cation channel which was highly similar to that found in the apical membrane of vestibular dark cells (Marcus et al., (1992) Am. J. Physiol. 262, C1423-C1429). The small nonselective cation channel was equally conductive (26.7 +/- 0.3 pS; N = 49) for K+, Na+, Rb+, Li+ and Cs+, 1.6 times more permeable to NH4+, but not permeable to Cl-, Ca2+, Ba2+ or N-methyl-D-glucamine. This channel yielded linear current-voltage relations which passed nearly through the origin (intercept: -2.2 +/- 0.4 mV, N = 49) when conductive monovalent cations were present on both sides of the membrane in equal concentrations. Channel activity required the presence of Ca2+ at the cytosolic face but not the extracellular (endolymphatic) face; there was essentially no activity for cytosolic Ca2+ less than or equal to 10(-7) M Ca2+ and full activity for greater than or equal to 10(-5) M. Cell-attached recordings had a conductance of 28.6 +/- 2.2 pS (N = 6) and a reversal voltage of -2.2 +/- 5.2 mV (N = 3) which was interpreted to reflect the intracellular potential of marginal cells under the present conditions. The three other types of channel were a Cl- channel (approximately 50 pS; N = 2), a maxi-K+ channel (approximately 230 pS; N = 1), and another large channel, probably cation nonselective (approximately 170 pS; N = 1). The 27 pS nonselective cation channel may be involved in K+ secretion and Na+ absorption under stimulated conditions which produce an elevated intracellular Ca2+; however, consideration of the apparent channel density in relation to the total transepithelial K+ flux suggests that these channels are not sufficient to account for K+ secretion.     

Altered Phenotype of the Vestibular Organ in GLAST-1 Null Mice

Various studies point to a crucial role of the high-affinity sodium-coupled glutamate aspartate transporter GLAST-1 for modulation of excitatory transmission as shown in the retina and the CNS. While 2–4-month-old GLAST-1 null mice did not show any functional vestibular abnormality, we observed profound circling behavior in older (7 months) animals lacking GLAST-1. An unchanged total number of otoferlin-positive vestibular hair cells (VHCs), similar ribbon numbers in VHCs, and an unchanged VGLUT3 expression in type II VHCs were detected in GLAST-1 null compared to wild-type mice. A partial loss of supporting cells and an apparent decline of a voltage-gated channel potassium subunit (KCNQ4) was observed in postsynaptic calyceal afferents contacting type I VHCs, together with a reduction of neurofilament- (NF200-) and vesicular glutamate transporter 1- (VGLUT1-) positive calyces in GLAST-1 null mice. Taken together, GLAST-1 deletion appeared to preferentially affect the maintenance of a normal postsynaptic/neuronal phenotype, evident only with increasing age.     

High potassium concentrations protect inner and outer hair cells in the newborn rat culture from ischemia-induced damage

Several studies indicate that an increase in the extracellular potassium (K+) concentration is a factor exerting a damaging effect on cochlear hair cells (HCs). The present study was designed to examine the effects of high extracellular K+ concentrations on the HCs under normoxic and ischemic conditions. Organotypic cultures of the organ of Corti of newborn rats were exposed to normoxia and ischemia at K+ concentrations of 5-70 mM in artificial perilymph for 3-4h. The number of IHCs and OHCs in the apical, medial and basal parts of the cochlea were counted 24h later. The work resulted in two main findings: (1) extracellular K+ concentrations of 30-70 mM had no effect on the HCs under normoxic conditions; (2) under ischemic conditions, a clear HC loss, mainly in the medial and basal cochlear parts, was observed at 5 mM K+ as previously reported. In contrast, a high extracellular K+ concentration strongly attenuated the HC loss. This effect nearly completely disappeared by the addition of both eosin, an inhibitor of the plasma membrane calcium ATPase (PMCA), and linopirdine, an inhibitor of the KCNQ4 channel, indicating that a normal activity of the PMCA and the KCNQ4 channels are key factors for HC survival under ischemia and depolarizing conditions.     

Functional aspects of the vestibular dark cells in the guinea pig: morphological investigation using ruthenium red staining technique.

The functional significance of the vestibular dark cells in the guinea pig was investigated using ruthenium red staining technique. The apical cell surface of the dark cell was covered by the fuzzy layer of the glycocalyx, which could be responsible for water and ion transport across the apical plasma membrane. It has been suggested that the otoconia have been at first demineralized by an unknown mechanism when they are attached to the dark cell surface, and the mucopolysaccharide and mucoproteins in the otoconial matrix are then absorbed by active pinocytosis in the dark cells. These functions may be closely related to the possible regulation of the calcium ion in the vestibular endolymph.     

Immunolocalization of ClC-K chloride channel in strial marginal cells and vestibular dark cells

Secretion of K⁺ into endolymph depends on a particular constellation of ion transport proteins in the apical and basolateral membranes of strial marginal cells and vestibular dark cells. One fundamental component is the large chloride conductance of the basolateral membrane, which recycles chloride taken up by the Na⁺-K⁺-Cl⁻ cotransporter in the same membrane. Evidence has been reported recently that supports ClC-K, a channel subunit previously thought to be specific to the kidney, as being the molecular entity underlying this conductance. We have isolated protein from the gerbil kidney, stria vascularis and vestibular labyrinth and found by Western blot analysis a 60 kDa band, a 48 kDa band and 54 and 70 kDa bands, respectively, specifically labeled by ClC-K antibody. Subsequent immunohistochemical observations of the inner ear tissues with a confocal microscope on fluorescently labeled tissue sections showed the staining to be restricted to the basolateral region of strial marginal cells and vestibular dark cells. The cochlear staining was distinct from the distribution of the Kir4.1 (KCNJ10) K⁺ channel, known to be present only in strial intermediate cells. These findings support the contention that ClC-K is an important component of the basolateral Cl⁻ conductance that participates in K⁺ secretion by these epithelia.     

Ultracytochemical study of Ouabain-sensitive, potassium-dependent p-nitrophenylphosphatase activity in the inner ear of the squirrel monkey

The localization of ouabain-sensitive, K(+)-dependent p-nitrophenylphosphatase (K(+)-NPPase) activity of the Na(+),K(+)-ATPase complex was studied ultracytochemically in the squirrel monkey inner ear. In the stria vascularis the reaction products showing K(+)-NPPase activity were limited to the cytoplasmic side of the plasmalemmal infoldings of the marginal cells. In the spiral prominence, a weak reaction was also found on the cytoplasmic process of the stromal cell, while no or little reaction was detected on the spiral prominence epithelium. In the dark cells of vestibular labyrinth the reaction products were observed on the basolateral interdigitation of the plasmalemma. In contrast, no reaction was observed on the apical cell surface. K(+)-NPPase activity was most intense in the strial marginal cell, followed by the dark cell of the ampulla and the utricle. The present results revealed that the dark cells in the vestibular labyrinth are involved in endolymph homeostasis.     

Ultracytochemical study of ouabain-sensitive, potassium-dependent p-nitrophenylphosphatase activity in the inner ear of the squirrel monkey

The localization of ouabain-sensitive, K+-dependent p-nitrophenylphosphatase (K+-NPPase) activity of the Na+, K+-ATPase complex was studied ultracytochemically in the squirrel monkey inner ear. In the stria vascularis the reaction products showing K+-NPPase activity were limited to the cytoplasmic side of the plasmalemmal infoldings of the marginal cells. In the spiral prominence, a weak reaction was also found on the cytoplasmic process of the stromal cell, while no or little reaction was detected on the spiral prominence epithelium. In the dark cells of vestibular labyrinth the reaction products were observed on the basolateral interdigitation of the plasmalemma. In contrast, no reaction was observed on the apical cell surface. K+-NPPase activity was most intense in the strial marginal cell, followed by the dark cell of the ampulla and the utricle. The present results revealed that the dark cells in the vestibular labyrinth are involved in endolymph homeostasis.     

Distribution of two-pore-domain potassium channels in the adult rat vestibular periphery

Constitutively active background or "leak" two-pore-domain potassium (K(+)) channels (Kcnk family), as defined by lack of voltage and time dependency are central to electrical excitability of cells by controlling resting membrane potential and membrane resistance. Inhibition of these channels by several neurotransmitters, e.g. glutamate, or acetylcholine, induces membrane depolarization and subsequent action potential firing as well as increases membrane resistance amplifying responses to synaptic inputs. In contrast, their opening contributes to hyperpolarization. Because of their central role in determining cellular excitability and response to synaptic stimulation, these channels likely play a role in the differential effects of vestibular efferent neurons on afferent discharge. Microarray data from previous experiments showed Kcnk 1, 2, 3, 6, 12 and 1 5 mRNA in Scarpa's ganglia. Real-time RT-PCR showed Kcnk 1, 2, 3, 6, 12 and 15 mRNA expression in Scarpa's ganglia and Kcnk 1, 2, 3, 6, 12 but not 15 mRNA expression in the crista ampullaris. We studied the distribution of two-pore-domain potassium channels K(2P)1.1, 2.1, 3.1 and 6.1 like immunoreactivity (corresponding to Kcnk genes 1, 2, 3 and 6) in the vestibular periphery. K(2P)1.1 (TWIK 1) immunoreactivity was detected along nerve terminals, supporting cells and blood vessels of the crista ampullaris and in the cytoplasm of neurons of the Scarpa's ganglia. K(2P)2.1 (TREK 1) immunoreactivity was detected in nerve terminals and transitional cells of the crista ampullaris, in the vestibular dark cells and in neuronal fibers and somata of neurons of Scarpa's ganglia. K(2P)3.1 (TASK 1) immunoreactivity was detected in supporting cells and transitional cells of the crista ampullaris, in vestibular dark cells and in neuron cytoplasm within Scarpa's ganglia. K(2P)6.1 (TWIK 2) immunoreactivity was detected in nerve terminals, blood vessels hair cells and transitional cells of the crista ampullaris and in the somata and neuron fibers of Scarpa's ganglia.     

Cytochemical studies of Ca++-ATPase activity in the vestibular epithelia of the guinea pig

We used ultracytochemistry to examine Ca++-ATPase activity in the vestibular epithelia of the guinea pig. Many reaction products were found along the basolateral plasma membrane of the vestibular dark cell. There were also marked reaction deposits on the apical and lateral cell membranes of the transitional cells, and the utricular and saccular wall cells. Both sensory and supporting cells showed Ca++-ATPase activity along their ciliary membrane and apical-lateral cell surfaces. Our findings indicate that the Ca++-ATPase activity found on the plasma membrane is closely related to Ca++-transport across the plasma membrane. When either Ca++ or ATP was omitted from the incubation medium, enzyme activity (as seen by the staining reaction present) was completely abolished. Our present results suggest that Ca++-ATPase located in the vestibular epithelia plays a significant role in the regulation of the Ca++-concentration in the vestibular endolymph.     

Secretion of endolymph by the isolated frog semicircular canal.

Secretion of endolymph is localized in some structures of the inner ear, namely the stria vascularis in the cochlea and the dark cells in the vestibule and in the lower vertebrate inner ear. In isolated semicircular canal it is possible to study separately the endolymphatic composition in the ampulla, which contains the dark cells, and in its non-ampullar part, which is devoid of these cells. Further, in vitro preparation of the semicircular canal provides access to both faces of the epithelium so that different agents can be applied separately to the apical or to the basolateral membranes of the epithelium. In this structure, the following results were obtained: i) in vitro, the semicircular canal secreted a K-rich, positively polarized fluid; ii) this fluid was secreted only in the ampulla of the semicircular canal; iii) the secretion of endolymph was dependent on basolateral Na+, K(+)-ATPase, inhibited by ouabain, and basolateral Na-K-Cl co-transporter, inhibited by bumetanide; iv) approximately 60% of luminal Na absorption occurred across a luminal Na channel inhibited by amiloride; v) the permeability of the paracellular pathway of the semicircular canal epithelium was 7.10(-7) cm/s. These results indicate that endolymph secretion involves basolateral Na+, K(+)-ATPase and Na-K-Cl co-transporter. An Na channel has been shown at the apical membrane.     

The effect of spatial adaptation on auditory motion processing

Na(+) concentrations in endolymph must be controlled to maintain hair cell function since the transduction channels of hair cells are cation-permeable, but not K(+)-selective. Flooding or fluctuations of the hair cell cytosol with Na(+) would be expected to lead to cellular dysfunction, hearing loss and vertigo. This review briefly describes cellular mechanisms known to be responsible for Na(+) homeostasis in each compartment of the inner ear, including the cochlea, saccule, semicircular canals and endolymphatic sac. The influx of Na(+) into endolymph of each of the organs is likely via passive diffusion, but these pathways have not yet been identified or characterized. Na(+) absorption is controlled by gate-keeper channels in the apical (endolymphatic) membrane of the transporting cells. Highly Na(+)-selective epithelial sodium channels (ENaCs) control absorption by Reissner's membrane, saccular extramacular epithelium, semicircular canal duct epithelium and endolymphatic sac. ENaC activity is controlled by a number of signal pathways, but most notably by genomic regulation of channel numbers in the membrane via glucocorticoid signaling. Non-selective cation channels in the apical membrane of outer sulcus epithelial cells and vestibular transitional cells mediate Na(+) and parasensory K(+) absorption. The K(+)-mediated transduction current in hair cells is also accompanied by a Na(+) flux since the transduction channels are non-selective cation channels. Cation absorption by all of these cells is regulated by extracellular ATP via apical non-selective cation channels (P2X receptors). The heterogeneous population of epithelial cells in the endolymphatic sac is thought to have multiple absorptive pathways for Na(+) with regulatory pathways that include glucocorticoids and purinergic agonists.     

Sodium- and potassium-activated ATPase in the mammalian vestibular system

Autoradiographic and cytochemical procedures were employed to determine the cellular distribution of the Na,K-ATPase enzyme in the mammalian vestibular system. A light-microscope survey of vestibular tissues incubated with [(3)H]ouabain shows high densities of ouabain binding sites within the dark cell epithelium (DC) of the ampullae of the semi-circular canals, and to a lesser extent, the DC of the utricular macula. A moderate number of binding sites was found in nerve fibers penetrating the connective tissue beneath the sensory epithelium (SE) of the ampullae and the maculae. A small number of binding sites is distributed in the deep portion of the SE, both in the ampullae and in the maculae. These latter binding sites seem to be associated with nerve terminals and receptor cells. At the ultrastructural level, the vestibular dark cells exhibit extensive basolateral membrane infolding, a morphological hallmark of cells engaged in trans-epithelial ion transport. The cytochemical reaction product is K(+)-dependent, ouabain inhibitable, and is restricted to the basolateral membrane extensions, with little or no product on the luminal membrane. The extent of membrane infolding in dark cells of the utricle is less pronounced than that of the ampullar dark cells and the intensity of the cytochemical reaction appears to correlate with the extent of membrane infolding. The results support the widely held hypothesis that the vestibular dark cells play a role in endolymph production. They also suggest that the vestibular sensory epithelia may be a site of ion exchange.