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 (ENaC) 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. Nonselective 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 nonselective cation channels. Cation absorption by all of these cells is regulated by extracellular ATP via apical nonselective 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.
inner ear; sodium homeostasis; epithelial sodium channel; Meniere’s disease
Slc26a4Δ/Δ mice are deaf, develop an enlarged membranous labyrinth, and thereby largely resemble the human phenotype where mutations of SLC26A4 cause an enlarged vestibular aqueduct and sensorineural hearing loss. The enlargement is likely caused by abnormal ion and fluid transport during the time of embryonic development, however, neither the mechanisms of ion transport nor the ionic composition of the luminal fluid during this time of development are known. Here we determine the ionic composition of inner ear fluids at the time at which the enlargement develops and the onset of expression of selected ion transporters. Concentrations of Na+ and K+ were measured with double-barreled ion-selective electrodes in the cochlea and the endolymphatic sac of Slc26a4Δ/+, which develop normal hearing, and of Slc26a4Δ/Δ mice, which fail to develop hearing. The expression of specific ion transporters was examined by quantitative RT-PCR and immunohistochemistry. High Na+ (∼141 mM) and low K+ concentrations (∼11 mM) were found at embryonic day (E) 16.5 in cochlear endolymph of Slc26a4Δ/+ and Slc26a4Δ/Δ mice. Shortly before birth the K+ concentration began to rise. Immediately after birth (postnatal day 0), the Na+ and K+ concentrations in cochlear endolymph were each ∼80 mM. In Slc26a4Δ/Δ mice, the rise in the K+ concentration occurred with a ∼3 day delay. K+ concentrations were also found to be low (∼15 mM) in the embryonic endolymphatic sac. The onset of expression of the K+ channel KCNQ1 and the Na+/2Cl−/K+ cotransporter SLC12A2 occurred in the cochlea at E19.5 in Slc26a4Δ/+ and Slc26a4Δ/Δ mice. These data demonstrate that endolymph, at the time at which the enlargement develops, is a Na+-rich fluid, which transitions into a K+-rich fluid before birth. The data suggest that the endolymphatic enlargement caused by a loss of Slc26a4 is a consequence of disrupted Na+ transport.
The vestibular system controls the ion composition of its luminal fluid through several epithelial cell transport mechanisms under hormonal regulation. The semicircular canal duct (SCCD) epithelium has been shown to secrete Cl- under β2-adrenergic stimulation. In the current study, we sought to determine the ion transporters involved in Cl- secretion and whether secretion is regulated by PKA and glucocorticoids.
Short circuit current (Isc) from rat SCCD epithelia demonstrated stimulation by forskolin (EC50: 0.8 μM), 8-Br-cAMP (EC50: 180 μM), 8-pCPT-cAMP (100 μM), IBMX (250 μM), and RO-20-1724 (100 μM). The PKA activator N6-BNZ-cAMP (0.1, 0.3 & 1 mM) also stimulated Isc. Partial inhibition of stimulated Isc individually by bumetanide (10 & 50 μM), and [(dihydroindenyl)oxy]alkanoic acid (DIOA, 100 μM) were additive and complete. Stimulated Isc was also partially inhibited by CFTRinh-172 (5 & 30 μM), flufenamic acid (5 μM) and diphenylamine-2,2′-dicarboxylic acid (DPC; 1 mM). Native canals of CFTR+/− mice showed a stimulation of Isc from isoproterenol and forskolin+IBMX but not in the presence of both bumetanide and DIOA, while canals from CFTR−/− mice had no responses. Nonetheless, CFTR−/− mice showed no difference from CFTR+/− mice in their ability to balance (rota-rod). Stimulated Isc was greater after chronic incubation (24 hr) with the glucocorticoids dexamethasone (0.1 & 0.3 μM), prednisolone (0.3, 1 & 3 μM), hydrocortisone (0.01, 0.1 & 1 μM), and corticosterone (0.1 & 1 μM) and mineralocorticoid aldosterone (1 μM). Steroid action was blocked by mifepristone but not by spironolactone, indicating all the steroids activated the glucocorticoid, but not mineralocorticoid, receptor. Expression of transcripts for CFTR; for KCC1, KCC3a, KCC3b and KCC4, but not KCC2; for NKCC1 but not NKCC2 and for WNK1 but only very low WNK4 was determined.
These results are consistent with a model of Cl- secretion whereby Cl- is taken up across the basolateral membrane by a Na+-K+-2Cl- cotransporter (NKCC) and potentially another transporter, is secreted across the apical membrane via a Cl- channel, likely CFTR, and demonstrate the regulation of Cl- secretion by protein kinase A and glucocorticoids.
Chloride secretion; Rat; Knockout mouse; Primary culture; Epithelium; Inner ear; Bumetanide; DIOA; Glucocorticoid; NKCC; KCC
Sensory transduction in the cochlea depends on regulated ion secretion and absorption. Results of whole-organ experiments suggested that Reissner’s membrane may play a role in the control of luminal Cl−. We tested for the presence of Cl− transport pathways in isolated mouse Reissner’s membrane using whole-cell patch clamp recording and gene transcript analyses using RT-PCR. The current-voltage (I-V) relationship in the presence of symmetrical NMDG-Cl was strongly inward-rectifying at negative voltages, with a small outward current at positive voltages. The inward-rectifying component of the I-V curve had several properties similar to those of the ClC-2 Cl− channel. It was stimulated by extracellular acidity and inhibited by extracellular Cd2+, Zn2+, and intracellular ClC-2 antibody. Channel transcripts expressed include ClC-2, Slc26a7 and ClC-Ka, but not Cftr, ClC-1, ClCa1, ClCa2, ClCa3, ClCa4, Slc26a9, ClC-Kb, Best1, Best2, Best3 or the beta-subunit of ClC-K, barttin. ClC-2 is the only molecularly-identified channel present that is a strong inward rectifier. This study is the first report of conductive Cl− transport in epithelial cells of Reissner’s membrane and is consistent with an important role in endolymph anion homeostasis.
Cl− channel; epithelial transport; cochlea
Epithelial cells of the inner ear coordinate their ion transport activity through a number of mechanisms. One important mechanism is the autocrine and paracrine signaling among neighboring cells in the ear via nucleotides, such as adenosine, ATP and UTP. This review summarizes observations on the release, detection and degradation of nucleotides by epithelial cells of the inner ear. Purinergic signaling is thought to be important for endolymph ion homeostasis and for protection from over stimulation.
cochlea; vestibular labyrinth; purinergic receptor; stria vascularis; marginal cell; vestibular dark cell; nucleotide release; ATP; UTP; ecto-nucleotidase
Purinergic receptors have been found to modulate ion transport in several types of epithelial cells as well as excitable cells. It was of interest to determine whether vestibular dark cells and strial marginal cells contain purinergic receptors in either the apicalor basolateral membrane which modulate transepithelial ion transport. Vestibular dark cell and strial marginal cell epithelia were mounted in a micro-Ussing chamber for the measurement of the transepithelial voltage and resistance from which the equivalent short circuit current (Isc) was obtained. The apical and basolateral sides were independently perfused with adenosine and adenosine 5′-triphosphate (ATP). Adenosine (10−5 M) had no effect on Isc at either the apical or basolateral side of vestibular dark cells and strial marginal cells, suggesting either the absence of P1 receptors or the absence of coupling of P1 receptors to vectorial ion transport by these epithelia. Apical perfusion of ATP (10−8 to 10−4 M) caused a decrease in Isc of both vestibular dark cells and strial marginal cells. Apical perfusion of the nucleotides uridine 5′-triphosphate (UTP), 2-methylthioadenosine triphosphate (2-meS-ATP), adenosine 5′-O-(3-thiotriphosphate) (ATPγS) and α,β-methylene adenosine 5′-triphosphate (α,β-meth-ATP) caused qualitatively similar responses with different magnitudes of response. The sequence of the magnitude of response of each compound at 10−6 or 10−5 M was assessed from the fractional change of Isc. The sequence for vestibular dark cells was UTP = ATP = ATPγS ≫ 2-meS-ATP > α,β-meth-ATP, and for strial marginal cells it was UTP = ATP ≫ 2-meS-ATP, corresponding to the sequence for the P2U receptor. The effect of agonist on the apical membrane was reduced by the antagonist 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS) but not cibacron blue or suramin. DIDS in the absence of exogenous purinergic agonist caused a sustained increase in Isc. The effect of ATP on the apical membrane was greater in the absence of divalent cations. Basolateral perfusion of ATP led to a biphasic response of Isc in vestibular dark cell and strial marginal cell epithelia, consisting of an initial rapid increase followed by a slower decrease. Perfusion of the perilymphatic surface of the stria vascularis (basal cell layer) with ATP had no acute effect on Isc. The initial increase of Isc in vestibular dark cell epithelium during basolateral perfusion had a sequence of 2-meS-ATP > ATP ≫ UTP = α,β-meth-ATP = ATPγS, corresponding to the sequence for the P2Y receptor. Subsequently, the agonists caused a sustained decrease in Isc with a sequence of ATPγS > 2-meS-ATP > ATP > UTP >α,β-meth-ATP. This sequence is most simply interpreted as the result of the coexistence of P2U and P2Y receptors in the basolateral membrane. Both the increase and decrease of Isc by ATP at the basolateral membrane were reduced by the antagonist suramin. These findings provide evidence for the regulation of transepithelial ion transport by P2U receptors in the apical membrane and by coexisting P2U and P2Y receptors in the basolateral membrane of K+-secretory epithelial cells in the inner ear and are consistent with the hypothesis that the apical receptors are part of an autocrine negative feedback system in these cells.
P2U receptor; P2 agonists; adenosine; DIDS; cibacron blue; suramin; reactive blue 2
Strial marginal cells (SMC) and vestibular dark cells (VDC) are known to secrete K+ into endolymph. Slowly-activating, voltage-dependent K+ channels (KCNQ1/KCNE1; IsK; min K) have been identified in the apical membrane of these cells. Several experimental maneuvers known to increase or decrease transepithelial K+ secretion have been found in VDC to change the current through these channels in the same ways. In both SMC and VDC the kinetics of activation and deactivation resemble those of the IsK channel exogenously expressed in Xenopus oocytes and endogenous to heart myocytes. The present study sought evidence that this current is indeed carried by IsK channels and that this current is the basis for transepithelial K+ secretion. Both on-cell macro-patch recordings of the apical membrane and perforated-patch whole-cell recordings were made on SMC from gerbil in order to measure macroscopic cell currents. The on-cell current was found to 1) be K+-selective, 2) have a cation permeability sequence of K+ ~ Rb+ > Cs+ >> Li+ = Na+, 3) be activated with a time constant of 1764 ± 413 ms by voltage steps from 0 to +40 mV, 4) be deactivated with a time constant of 324 ± 57 ms by voltage steps from 0 to -40 mV and 5) be reduced 84 ± 5% by bumetanide (10-5 M), an inhibitor of K+ secretion. The single-channel conductance of the apical currents in the homologous VDC was estimated by fluctuation analysis to be 1.6 pS. The potent inhibitor of IsK channels, chromanol 293B (10-5 M), reduced the whole-cell current in SMC by 72 ± 10 %. Clofilium (10-4 M), a putative IsK channel inhibitor known to have additional non-specific effects, led to a stimulation of both on-cell (by 598 ± 177%) and whole-cell (by 162 ± 18%) currents in gerbil SMC but to a decrease of whole-cell currents (by 39 ± 12%) in rat SMC. Taken together with other findings reviewed here, these results strongly argue that the slowly-activating, voltage-dependent conductance in the apical membrane of SMC is the IsK channel and provide additional evidence for the poor specificity of clofilium.
K+ channel; perforated-patch whole-cell voltage clamp; fluctuation analysis; KCNQ1/KCNE1 K channel; gerbil; rat
Vestibular dark cell epithelium was isolated from the semicircular canal of gerbils to test the proposal that the sulfhydryl alkylating agent N-ethylmaleimide (NEM) inhibits K+ secretion by this tissue and does so by reacting with a site in or near the apical membrane. Dark cell epithelium was mounted in a micro-Ussing chamber for measurements of transepithelial voltage (Vt) and resistance (Rt) or in a perfused bath on the stage of a microscope for measurement of cell height as an index of cell volume. Perfusion of the apical or basolateral side with 10−3 M NEM caused an increase in Vt superimposed upon a slower decrease of Vt, resulting in a triphasic response. There were only small changes in Rt. Under this condition, Vt is proportional to short circuit current and to K+ secretion. Both the stimulatory and the inhibitory responses of Vt were dose-dependent between 10−6 and 10−3 M NEM and the inhibition was irreversible. The specificity of the reaction of NEM with sulfhydryl groups was confirmed by the use of the reducing agent dithiothreitol (DTT). Perfusion of 5×10−4 M DTT on the apical side caused no significant changes in Vt but completely prevented both stimulation and inhibition of Vt by NEM (10−3 M). The amplitudes of the stimulation and the inhibition of Vt were greater for basolateral than for apical perfusion of NEM. Similarly, the response times for each effect were faster from the basolateral side, suggesting that the primary sites of action are at or near the basolateral membrane. The site of action of NEM was further explored by subjecting the tissue to a membrane-impermeant sulfhydryl reagent, stilbenedisulfonate maleimide (SDM). Apical perfusion of 10−3 M SDM had no effect on Vt or Rt, whereas basolateral perfusion caused a reversible increase of Vt (5.2 ± 0.5 to initially 6.8 ± 0.5 mV which relaxed after 60 s to 5.8 ± 0.5 mV) and to an initial decrease in Rt by 4%. No inhibitory phase was observed. Elevation of basolateral [K+] from 3.6 to 25 mM is known to increase Vt and reduce Rt via direct stimulation of basolateral K+ uptake and indirect stimulation of the apical membrane conductance. Basolateral perfusion of 10−3 M NEM fully inhibited the increase of Vt due to 25 mM K+. Elevation of basolateral [K+] from 3.6 to 25 mM is known to increase reversibly cell volume. NEM was found to inhibit cell swelling in a dose-dependent manner but did not initially affect the rate of shrinking after K+-induced swelling, pointing to action only on basolateral transport pathways. The effects of NEM on K+-induced cell swelling were completely prevented by 5×10−4 M DTT, demonstrating that the inhibitory effect of NEM was on sulfhydryl groups. In contrast to interpretations of NEM action in frog semicircular canal, we have found that NEM appears to stimulate an ion transport process in mammalian dark cells at an extracellular site in the basolateral membrane and inhibits another ion transport process in the basolateral membrane at another site. Inhibition by NEM from the apical side occurs most likely by diffusion of the agent to a site at or near the cytosolic side of the basolateral membrane.
Vestibular labyrinth; epithelial transport; cell volume; dark cells; sulfhydryl reagents
Sodium absorption by semicircular canal duct (SCCD) epithelial cells is thought to contribute to the homeostasis of the volume of vestibular endolymph. It was previously shown that the epithelial cells could absorb Na+ under control of a glucocorticoid hormone (dexamethasone) and the absorptive transepithelial current was blocked by amiloride. The most commonly-observed target of amiloride is the epithelial sodium channel (ENaC), comprised of the three subunits α-, β- and γ-ENaC. However, other cation channels have also been observed to be sensitive in a similar concentration range. The aim of this study was to determine whether SCCD epithelial cells absorb only Na+ or also K+ through an amiloride-sensitive pathway. Parasensory K+ absorption could contribute to regulation of the transduction current through hair cells, as found to occur via vestibular transitional cells [S. H. Kim and D. C. Marcus. Regulation of sodium transport in the inner ear. Hear.Res. doi:10.1016/j.heares.2011.05.003, 2011].
We determined the molecular and functional expression of candidate cation channels with gene array (GEO GSE6197), whole-cell patch clamp and transepithelial recordings in primary cultures of rat SCCD. α-, β- and γ-ENaC were all previously reported as present. The selectivity of the amiloride-sensitive transepithelial and cell membrane currents was observed in Ussing chamber and whole-cell patch clamp recordings. The cell membrane currents were carried by Na+ but not K+, but the Na+ selectivity disappeared when the cells were cultured on impermeable supports. Transepithelial currents across SCCD were also carried exclusively by Na+.
These results are consistent with the amiloride-sensitive absorptive flux of SCCD mediated by a highly Na+-selective channel, likely αβγ-ENaC. These epithelial cells therefore absorb only Na+ via the amiloride-sensitive pathway and do not provide a parasensory K+ efflux from the canals via this pathway. The results further provide caution to the culture of epithelial cells on impermeable surfaces.
Sodium absorption by Reissner's membrane is thought to contribute to the homeostasis of the volume of cochlear endolymph. It was previously shown that the absorptive transepithelial current was blocked by amiloride and benzamil. The most commonly-observed target of these drugs is the epithelial sodium channel (ENaC), which is composed of the three subunits α-,β- and γ-ENaC. However, other less-selective cation channels have also been observed to be sensitive to benzamil and amiloride. The aim of this study was to determine whether Reissner's membrane epithelial cells could support parasensory K+ absorption via amiloride- and benzamil-sensitive electrogenic pathways.
We determined the molecular and functional expression of candidate cation channels with gene array (GEO GSE6196), RT-PCR, and whole-cell patch clamp. Transcript expression analysis of Reissner's membrane detected no amiloride-sensitive acid-sensing ion channels (ASIC1a, ASIC2a, ASIC2b) nor amiloride-sensitive cyclic-nucleotide gated channels (CNGA1, CNGA2, CNGA4, CNGB3). By contrast, α-,β- and γ-ENaC were all previously reported as present in Reissner's membrane. The selectivity of the benzamil-sensitive cation currents was observed in whole-cell patch clamp recordings under Cl--free conditions where cations were the only permeant species. The currents were carried by Na+ but not K+, and the permeability of Li+ was greater than that of Na+ in Reissner's membrane. Complete replacement of bath Na+ with the inpermeable cation NMDG+ led to the same inward current as with benzamil in a Na+ bath.
These results are consistent with the amiloride/benzamil-sensitive absorptive flux of Reissner's membrane mediated by a highly Na+-selective channel that has several key characteristics in common with αβγ-ENaC. The amiloride-sensitive pathway therefore absorbs only Na+ in this epithelium and does not provide a parasensory K+ efflux route from scala media.
The ionic composition of the luminal fluid in the vestibular labyrinth is maintained within tight limits by the many types of epithelial cells bounding the lumen. Regulatory mechanisms include systemic, paracrine and autocrine hormones along with their associated intracellular signal pathways. The epithelium lining the semicircular canal duct (SCCD) is a tissue that is known to absorb sodium and calcium and to secrete chloride.
Transport function was assessed by measurements of short circuit current (Isc) and gene transcript expression was evaluated by microarray. Neither ATP nor UTP (100 microM) on the apical side of the epithelium had any effect on Isc. By contrast, basolateral ATP transiently increased Isc and transepithelial resistance dropped significantly after basolateral ATP and UTP. P2Y2 was the sole UTP-sensitive purinergic receptor expressed. Isc was reduced by 42%, 50% and 63% after knockdown of α-ENaC, stimulation of PKC and inhibition of PI3-K, while the latter two increased the transepithelial resistance. PKCdelta, PKCgamma and PI3-K were found to be expressed.
These observations demonstrate that ion transport by the SCCD is regulated by P2Y2 purinergic receptors on the basolateral membrane that may respond to systemic or local agonists, such as ATP and/or UTP. The sodium absorption from endolymph mediated by ENaC in SCCD is regulated by signal pathways that include the kinases PKC and PI3-K. These three newly-identified regulatory components may prove to be valuable drug targets in the control of pathologic vestibular conditions involving dysfunction of transport homeostasis in the ear, such as Meniere's disease.
The low luminal Ca2+ concentration of mammalian endolymph in the inner ear is required for normal hearing and balance. We recently reported the expression of mRNA for a Ca2+-absorptive transport system in primary cultures of semicircular canal duct (SCCD) epithelium.
We now identify this system in native vestibular and cochlear tissues by qRT-PCR, immunoblots and confocal immunolocalization. Transcripts were found and quantified for several isoforms of epithelial calcium channels (TRPV5, TRPV6), calcium buffer proteins (calbindin-D9K, calbindin-D28K), sodium-calcium exchangers (NCX1, NCX2, NCX3) and plasma membrane Ca2+-ATPase (PMCA1, PMCA2, PMCA3, and PMCA4) in native SCCD, cochlear lateral wall (LW) and stria vascularis (SV) of adult rat as well as Ca2+ channels in neonatal SCCD. All components were expressed except TRPV6 in SV and PMCA2 in SCCD. 1,25-(OH)2vitamin D3 (VitD) significantly up-regulated transcripts of TRPV5 in SCCD, calbindin-D9K in SCCD and LW, NCX2 in LW, while PMCA4 in SCCD and PMCA3 in LW were down-regulated. The expression of TRPV5 relative to TRPV6 was in the sequence SV > Neonatal SCCD > Adult SCCD > LW > primary culture SCCD. Expression of TRPV5 protein from primary culture of SCCD did not increase significantly when cells were incubated with VitD (1.2 times control; P > 0.05). Immunolocalization showed the distribution of TRPV5 and TRPV6. TRPV5 was found near the apical membrane of strial marginal cells and both TRPV5 and TRPV6 in outer and inner sulcus cells of the cochlea and in the SCCD of the vestibular system.
These findings demonstrate for the first time the expression of a complete Ca2+ absorptive system in native cochlear and vestibular tissues. Regulation by vitamin D remains equivocal since the results support the regulation of this system at the transcript level but evidence for control of the TRPV5 channel protein was lacking.
Hereditary hearing loss is one of the most common birth defects, yet the majority of genes required for audition is thought to remain unidentified. Ethylnitrosourea (ENU)–mutagenesis has been a valuable approach for generating new animal models of deafness and discovering previously unrecognized gene functions. Here we report on the characterization of a new ENU–induced mouse mutant (nmf329) that exhibits recessively inherited deafness. We found a widespread loss of sensory hair cells in the hearing organs of nmf329 mice after the second week of life. Positional cloning revealed that the nmf329 strain carries a missense mutation in the claudin-9 gene, which encodes a tight junction protein with unknown biological function. In an epithelial cell line, heterologous expression of wild-type claudin-9 reduced the paracellular permeability to Na+ and K+, and the nmf329 mutation eliminated this ion barrier function without affecting the plasma membrane localization of claudin-9. In the nmf329 mouse line, the perilymphatic K+ concentration was found to be elevated, suggesting that the cochlear tight junctions were dysfunctional. Furthermore, the hair-cell loss in the claudin-9–defective cochlea was rescued in vitro when the explanted hearing organs were cultured in a low-K+ milieu and in vivo when the endocochlear K+-driving force was diminished by deletion of the pou3f4 gene. Overall, our data indicate that claudin-9 is required for the preservation of sensory cells in the hearing organ because claudin-9–defective tight junctions fail to shield the basolateral side of hair cells from the K+-rich endolymph. In the tight-junction complexes of hair cells, claudin-9 is localized specifically to a subdomain that is underneath more apical tight-junction strands formed by other claudins. Thus, the analysis of claudin-9 mutant mice suggests that even the deeper (subapical) tight-junction strands have biologically important ion barrier function.
Hereditary deafness is a common birth defect in the human population, yet the majority of genes required for audition is thought to be unidentified. Genetic approaches in the mouse have greatly contributed to our understanding of the molecular mechanisms that underlie hearing. Random mutagenesis of mice, identification of deaf mutants, and subsequent analysis of the deafness-causing gene defects has led to the discovery of several previously unrecognized gene functions. Here, we report on the characterization of a new mutant mouse line (nmf329) that exhibits profound hearing loss and loss of sensory cells in the auditory organ. Genetic analysis reveals that these animals carry a mutation in the claudin-9 gene, which encodes a protein with hitherto unknown biological function. We have found that normal claudin-9—but not a mutant form—inhibits the paracellular movement of certain ions. The lack of the claudin-9 ion barrier in the inner ear leads to changes in ionic conditions that can account for the loss of sensory cells in the mutant mice. Within the cell–cell junctions, the claudin-9 layer is located basal to those of other claudins. Thus, our analysis of claudin-9–deficient animals suggests that even the deeper layers of claudins have important ion barrier function.
The low Ca2+ concentration of mammalian endolymph in the inner ear is required for normal hearing and balance. We reported [Yamauchi et al. Biochem Biophys Res Commun, 2005] that the epithelial Ca2+ channels TRPV5 and TRPV6 are expressed in the vestibular system and that TRPV5 expression is stimulated by 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), as also reported in kidney. TRPV5/6 channels are known to be inhibited by extracellular acidic pH. Endolymphatic pH, [Ca2+] and transepithelial potential of the utricle (UP) were measured in Cl-/
HCO3− exchanger pendrin (SLC26A4) knockout mice in vivo. Slc26a4-/- mice exhibit reduced pH and UP and increased [Ca2+]. Monolayers of primary cultures of rat semicircular canal duct (SCCD) cells were grown on permeable supports and cellular uptake of 45Ca2+ was measured individually from the apical and basolateral sides. Net uptake of 45Ca2+ was greater after incubation with 1,25(OH)2D3. Net 45Ca2+ absorption was dramatically inhibited by low apical pH and was stimulated by apical alkaline pH. Gadolinium, lanthanum and ruthenium red reduced apical uptake. These observations support the notion that one aspect of vestibular dysfunction in Pendred syndrome is a pathological elevation of endolymphatic [Ca2+] due to luminal acidification and consequent inhibition of TRPV5/6-mediated Ca2+ absorption.
Epithelial Ca Channel; vitamin D; SLC26A4; HCO3- secretion; TRPV5; TRPV6
Pendred syndrome, characterized by childhood deafness and postpuberty goiter, is caused by mutations of SLC26A4, which codes for the anion exchanger pendrin. The goal of the present study was to determine how loss of pendrin leads to hair cell degeneration and deafness. We evaluated pendrin function by ratiometric microfluorometry, hearing by auditory brain stem recordings, and expression of K+ and Ca2+ channels by confocal immunohistochemistry. Cochlear pH and Ca2+ concentrations and endocochlear potential (EP) were measured with double-barreled ion-selective microelectrodes. Pendrin in the cochlea was characterized as a formate-permeable and DIDS-sensitive anion exchanger that is likely to mediate
HCO3− secretion into endolymph. Hence endolymph in Slc26a4+/− mice was more alkaline than perilymph, and the loss of pendrin in Slc26a4−/− mice led to an acidification of endolymph. The stria vascularis of Slc26a4−/− mice expressed the K+ channel Kcnj10 and generated a small endocochlear potential before the normal onset of hearing at postnatal day 12. This small potential and the expression of Kcnj10 were lost during further development, and Slc26a4−/− mice did not acquire hearing. Endolymphatic acidification may be responsible for inhibition of Ca2+ reabsorption from endolymph via the acid-sensitive epithelial Ca2+ channels Trpv5 and Trpv6. Hence the endolymphatic Ca2+ concentration was found elevated in Slc26a4−/− mice. This elevation may inhibit sensory transduction necessary for hearing and promote the degeneration of the sensory hair cells. Degeneration of the hair cells closes a window of opportunity to restore the normal development of hearing in Slc26a4−/− mice and possibly human patients suffering from Pendred syndrome.
pendrin; stria vascularis; Slc26a4; Kcnj10; Trpv5
Mutations of SLC26A4 are a common cause of human hearing loss associated with enlargement of the vestibular aqueduct. SLC26A4 encodes pendrin, an anion exchanger expressed in a variety of epithelial cells in the cochlea, the vestibular labyrinth and the endolymphatic sac. Slc26a4Δ/Δ mice are devoid of pendrin and develop a severe enlargement of the membranous labyrinth, fail to acquire hearing and balance, and thereby provide a model for the human phenotype. Here, we generated a transgenic mouse line that expresses human SLC26A4 controlled by the promoter of ATP6V1B1. Crossing this transgene into the Slc26a4Δ/Δ line restored protein expression of pendrin in the endolymphatic sac without inducing detectable expression in the cochlea or the vestibular sensory organs. The transgene prevented abnormal enlargement of the membranous labyrinth, restored a normal endocochlear potential, normal pH gradients between endolymph and perilymph in the cochlea, normal otoconia formation in the vestibular labyrinth and normal sensory functions of hearing and balance. Our study demonstrates that restoration of pendrin to the endolymphatic sac is sufficient to restore normal inner ear function. This finding in conjunction with our previous report that pendrin expression is required for embryonic development but not for the maintenance of hearing opens the prospect that a spatially and temporally limited therapy will restore normal hearing in human patients carrying a variety of mutations of SLC26A4.
Mutations of SLC26A4 are the most common cause for hearing loss associated with a swelling of the inner ear. This human disease is largely recapitulated in a mutant mouse model. Mutant mice lack Slc26a4 expression and their inner ears swell during embryonic development, which leads to failure of the cochlea and the vestibular organs resulting in deafness and loss of balance. SLC26A4 is normally found in the cochlea and vestibular organs of the inner ear as well as in the endolymphatic sac, which is a non-sensory part of the inner ear. The multitude of sites where SLC26A4 is located made the goal to restore function through restoration look futile, unless some sites were more important than others. Here, we generated a new mutant mouse that expresses SLC26A4 in the endolymphatic sac but not in the cochlea or the vestibular organs of the inner ear. Fantastically, this mouse did not develop the detrimental swelling of the inner ear and even more exciting, the mouse developed normal hearing and balance. Our study provides the proof-of-concept that a therapy aimed at repairing the endolymphatic sac during embryonic development is sufficient to restore a life-time of normal hearing and balance.
It was previously shown that K+ secretion by strial marginal cell epithelium is under the control of G-protein coupled receptors of the P2Y family in the apical membrane. Receptor activation by uracil nucleotides (P2Y2, P2Y4 or P2Y6) leads to a decrease in the electrogenic K+ secretion. The present study was conducted to determine the subtype of the functional purinergic receptor in gerbil stria vascularis, to test if receptor activation leads to elevation of intracellular [Ca2+] and to test if the response to these receptors undergoes desensitization.
The transepithelial short circuit current (Isc) represents electrogenic K+ secretion and was found to be decreased by uridine 5'-triphosphate (UTP), adenosine 5'-triphosphate (ATP) and diadenosine tetraphosphate (Ap4A) but not uridine 5'-diphosphate (UDP) at the apical membrane of marginal cells of the gerbil stria vascularis. The potencies of these agonists were consistent with rodent P2Y4 and P2Y2 but not P2Y6 receptors. Activation caused a biphasic increase in intracellular [Ca2+] that could be partially blocked by 2-aminoethoxy-diphenyl borate (2-APB), an inhibitor of the IP3 receptor and store-operated channels. Suramin (100 μM) did not inhibit the effect of UTP (1 μM). The ineffectiveness of suramin at the concentration used was consistent with P2Y4 but not P2Y2. Transcripts for both P2Y2 and P2Y4 were found in the stria vascularis. Sustained exposure to ATP or UTP for 15 min caused a depression of Isc that appeared to have two components but with apparently no chronic desensitization.
The results support the conclusion that regulation of K+ secretion across strial marginal cell epithelium occurs by P2Y4 receptors at the apical membrane. The apparent lack of desensitization of the response is consistent with two processes: a rapid-onset phosphorylation of KCNE1 channel subunit and a slower-onset of regulation by depletion of plasma membrane PIP2.
The CD-1 mouse strain is known to have early onset of hearing loss that is progressive with aging. We sought to determine whether a disturbance of K+ homeostasis and pathological changes in the cochlear lateral wall were involved in the age-related hearing loss (AHL) of CD-1 as compared to the CBA/CaJ strain which has minimal AHL. In the present study, the endocochlear potential (EP) and endolymphatic K+ concentration ([K+]e) were measured in both strains of mice with double-barrel microelectrodes at “young” (1–2 mo) and “old” (5–9 mo) ages. CBA/CaJ mice displayed no changes with aging in EP and [K+]e of the basal turn. In the apical turn, there was a small positive shift of the EP (10 mV) with aging under both normoxic and acute anoxic conditions (−EP), without any change of [K+]e. Further, there were no obvious pathological changes in the lateral wall of CBA/CaJ mice. By contrast, old CD-1 mice displayed a significantly reduced [K+]e by 30% in both basal and apical turns with no significant changes in normoxic EP. The −EP in the apical turn was significantly reduced in magnitude by 6 mV. A severe loss of cells with aging was observed in the region of type IV fibrocytes of the apical and basal turns and of type II fibrocytes in the basal turn. A complete degeneration of organ of Corti was also observed at the basal turn of old CD-1 mice, as well as a basalward decline of spiral ganglion neuron density. The pathological changes in spiral ligament of CD-1 mice were similar to those of an inbred mouse strain C57BL/6J that expresses an AHL gene (ahl) and might be a primary etiology of AHL of CD-1 mice. These findings have ramifications for our understanding of AHL and for interpretation of genetic mutations in a CD-1 background.
stria vascularis; endocochlear potential; cochlea; potassium concentration; histology; age-related hearing loss
Immunolocalization studies of mouse cochlea and vestibular end-organ were performed to study the expression pattern of pendrin, the protein encoded by the Pendred syndrome gene (PDS), in the inner ear. The protein was restricted to the areas composed of specialized epithelial cells thought to play a key role in regulating the composition and resorption of endolymph. In the cochlea, pendrin was abundant in the apical membrane of cells in the spiral prominence and outer sulcus cells (along with their root processes). In the vestibular end-organ, pendrin was found in the transitional cells of the cristae ampullaris, utriculi, and sacculi as well as in the apical membrane of cells in the endolymphatic sac. Pds-knockout (Pds−/−) mice were found to lack pendrin immunoreactivity in all of these locations. Histological studies revealed that the stria vascularis in Pds−/− mice was only two-thirds the thickness seen in wild-type mice, with the strial marginal cells showing irregular shapes and sizes. Functional studies were also performed to examine the role of pendrin in endolymph homeostasis. Using double-barreled electrodes placed in both the cochlea and the utricle, the endocochlear potential and endolymph potassium concentration were measured in wild-type and Pds−/− mice. Consistent with the altered strial morphology, the endocochlear potential in Pds−/− mice was near zero and did not change during anoxia. On the other hand, the endolymphatic potassium concentration in Pds−/− mice was near normal in the cochlea and utricle. Together, these results suggest that pendrin serves a key role in the functioning of the basal and/or intermediate cells of the stria vascularis to maintain the endocochlear potential, but not in the potassium secretory function of the marginal cells.
Pendred syndrome, a common autosomal-recessive disorder characterized by congenital deafness and goiter, is caused by mutations of SLC26A4, which codes for pendrin. We investigated the relationship between pendrin and deafness using mice that have (Slc26a4+/+) or lack a complete Slc26a4 gene (Slc26a4-/-).
Expression of pendrin and other proteins was determined by confocal immunocytochemistry. Expression of mRNA was determined by quantitative RT-PCR. The endocochlear potential and the endolymphatic K+ concentration were measured with double-barreled microelectrodes. Currents generated by the stria marginal cells were recorded with a vibrating probe. Tissue masses were evaluated by morphometric distance measurements and pigmentation was quantified by densitometry.
Pendrin was found in the cochlea in apical membranes of spiral prominence cells and spindle-shaped cells of stria vascularis, in outer sulcus and root cells. Endolymph volume in Slc26a4-/- mice was increased and tissue masses in areas normally occupied by type I and II fibrocytes were reduced. Slc26a4-/- mice lacked the endocochlear potential, which is generated across the basal cell barrier by the K+ channel KCNJ10 localized in intermediate cells. Stria vascularis was hyperpigmented, suggesting unalleviated free radical damage. The basal cell barrier appeared intact; intermediate cells and KCNJ10 mRNA were present but KCNJ10 protein was absent. Endolymphatic K+ concentrations were normal and membrane proteins necessary for K+ secretion were present, including the K+ channel KCNQ1 and KCNE1, Na+/2Cl-/K+ cotransporter SLC12A2 and the gap junction GJB2.
These observations demonstrate that pendrin dysfunction leads to a loss of KCNJ10 protein expression and a loss of the endocochlear potential, which may be the direct cause of deafness in Pendred syndrome.