Purinergic receptors protect the cochlea during high-intensity stimulation by providing a parallel shunt pathway through non-sensory neighboring epithelial cells for cation absorption. So far, there is no direct functional evidence for the presence and type/subunit of purinergic receptors in the utricle of the vestibular labyrinth. The goal of the present study was to investigate which purinergic receptors are expressed and carry cation-absorption currents in the utricular transitional cells and macula. Purinergic agonists induced cation-absorption currents with a potency order of ATP > bzATP = αβmeATP ≫ ADP = UTP = UDP. ATP and bzATP are full agonists, whereas αβmeATP is a partial agonist. ATP-induced currents were partially inhibited by 100 μM suramin, 10 μM pyridoxal-phosphate-6-azo-(benzene-2,4-disulfonic acid (PPADS), or 5 μM 5-(3-bromophenyl)-1,3-dihydro-2H-benzofuro[3,2-e]-1, 4-diazepin-2-one (5-BDBD), and almost completely blocked by 100 μM Gd3+ or by a combination of 10 μM PPADS and 5 μM 5-BDBD. Expression of the P2RX2 and P2RX4 receptor was detected by immunocytochemistry in transitional cells and macular supporting cells. This is the first study to demonstrate that ATP induces cation currents carried by a combination of P2RX2 and P2RX4 in utricular transitional and macular epithelial cells, and supporting the hypothesis that purinergic receptors protect utricular hair cells during elevated stimulus intensity levels.

The sensory functions of the auditory and vestibular peripheral organs (inner ear) depend on vigorous transepithelial ion transport processes under the control of hormones and other signal pathways. The most salient of these are secretion of K+ and absorption of Na+ and Ca2+ by the concerted action of multiple epithelial cell types. Much has been learned of the molecular bases and development of these processes, as well as the genetic basis of common cochlear and vestibular diseases. The salient aspects are concisely reviewed in this chapter.

Unipolar brush cells (UBCs) are excitatory granular layer interneurons in the vestibulocerebellum. Here we assessed motor coordination and balance to investigate if deletion of acid-sensing ion channel 5 (Asic5), which is richly expressed in type II UBCs, is sufficient to cause ataxia. The possible cellular mechanism underpinning ataxia in this global Asic5 knockout model was elaborated using brain slice electrophysiology. Asic5 deletion impaired motor performance and decreased intrinsic UBC excitability, reducing spontaneous action potential firing by slowing maximum depolarization rate. Reduced intrinsic excitability in UBCs was partially compensated by suppression of the magnitude and duration of delayed hyperpolarizing K + currents triggered by glutamate. Glutamate typically stimulates burst firing subsequent to this hyperpolarization in normal type II UBCs. Burst firing frequency was elevated in knockout type II UBCs because it was initiated from a more depolarized potential compared to normal cells. Findings indicate that Asic5 is important for type II UBC activity and that loss of Asic5 contributes to impaired movement, likely, at least in part, due to altered temporal processing of vestibular input.

â Claudin expression raw dataâ . This file contains the data points collected and analyzed in the text and figures. (XLSX 86 kb)

Claudins are major components of tight junctions, which form the paracellular barrier between the cochlear luminal and abluminal fluid compartments that supports the large transepithelial voltage difference and the large concentration differences of K+, Na+ and Ca2+ needed for normal cochlear function. Claudins are a family of more than 20 subtypes, but our knowledge about expression and localization of each subtype in the cochlea is limited.

The transduction of sound into nerve impulses requires an ionic environment that depends on a variety of ion transport processes in epithelial and endothelial cells of the cochlea. Specific ion transport functions occur in specific cell types that coordinate the production and maintenance of endolymph, which is the fluid in the lumen of the cochlear duct that supports the sensory transduction process. The critical nature of these ion transport processes is underscored by observations of hearing loss when ion transport mechanisms malfunction as a result of mutations, drug exposure, or hormonal imbalance. This chapter describes our basic understanding of salient ion transport processes and their regulation by hormones and other regulatory pathways.

Disturbance of acid–base balance in the inner ear is known to be associated with hearing loss in a number of conditions including genetic mutations and pharmacologic interventions. Several previous physiologic and immunohistochemical observations lead to proposals of the involvement of acid–base transporters in stria vascularis.

Several members of the SLC26 gene family have highly-restricted expression patterns in the auditory and vestibular periphery and mutations in mice of at least two of these (SLC26A4 and SLC26A5) lead to deficits in hearing and/or balance. A previous report pointed to SLC26A7 as a candidate gene important for cochlear function. In the present study, inner ears were assayed by immunostaining for Slc26a7 in neonatal and adult mice. Slc26a7 was detected in the basolateral membrane of Reissner's membrane epithelial cells but not neighboring cells, with an onset of expression at P5; gene knockout resulted in the absence of protein expression in Reissner's membrane. Whole-cell patch clamp recordings revealed anion currents and conductances that were elevated for NO3- over Cl- and inhibited by I- and NPPB. Elevated NO3- currents were absent in Slc26a7 knockout mice. There were, however, no major changes to hearing (auditory brainstem response) of knockout mice during early adult life under constitutive and noise exposure conditions. The lack of Slc26a7 protein expression found in the wild-type vestibular labyrinth was consistent with the observation of normal balance. We conclude that SLC26A7 participates in Cl- transport in Reissner's membrane epithelial cells, but that either other anion pathways, such as ClC-2, possibly substitute satisfactorily under the conditions tested or that Cl- conductance in these cells is not critical to cochlear function. The involvement of SLC26A7 in cellular pH regulation in other epithelial cells leaves open the possibility that SLC26A7 is needed in Reissner's membrane cells during local perturbations of pH.

Background 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 β 2 -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. Results Short circuit current ( I sc ) from rat SCCD epithelia demonstrated stimulation by forskolin (EC 50 : 0.8 μM), 8-Br-cAMP (EC 50 : 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 I sc . Partial inhibition of stimulated I sc individually by bumetanide (10 & 50 μM), and [(dihydroindenyl)oxy]alkanoic acid (DIOA, 100 μM) were additive and complete. Stimulated I sc was also partially inhibited by CFTR inh -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 I sc 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 I sc 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. Conclusions 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.

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.

Slc26a4 (Δ/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 Slc26a4 (Δ/+), which develop normal hearing, and of Slc26a4 (Δ/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 Slc26a4 (Δ/+) and Slc26a4 (Δ/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 Slc26a4 (Δ/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 Slc26a4 (Δ/+) and Slc26a4 (Δ/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.

Mutations of SLC26A4 are a common cause for deafness associated with enlarged vestibular aqueduct. SLC26A4 encodes pendrin, an anion exchanger expressed in a variety of epithelial cells in the cochlea and the vestibular labyrinth. Slc26a4Δ/Δ mice develop a severe enlargement of the membranous labyrinth, fail to acquire hearing and balance, and thereby provide a stark model for the human phenotype. Here we generated a transgenic mouse line that expresses SLC26A4 under the promoter of ATP6V1B1. Crossing this transgene into the Slc26a4Δ/Δ line restored the temporal and spatial expression pattern of pendrin in the endolymphatic sac without inducing expression in the cochlea or the vestibular sensory organs. The transgene prevented enlargement and restored normal hearing and balance. Our study demonstrates that a limited restoration of pendrin is sufficient to restore normal inner ear function. These data in conjunction with notion that pendrin is required for the development but not the maintenance of hearing (Choi et al., 2011) opens the prospect that a spatially and temporally limited therapy may restore a life‐time of normal hearing in Slc26a4Δ/Δ mice, and possibly humans with SLC26A4 mutations.Supported by NIH‐R01‐DC012151, NIH‐P20‐RR 17686, KINBRE, Kansas State University, ANR BLANC 2010‐R10164DD

Abstract Conclusion: Claudius' cells absorb Na+ through the amiloride-sensitive epithelial sodium channel (ENaC). Transepithelial ion transport through ENaC and possibly a Cl– secretory pathway is regulated by P2Y purinergic signaling. Objectives: The purpose of this study was to investigate ion transport in Claudius' cells and its purinergic regulation. Methods: Young adult Sprague–Dawley rats and gerbils were studied. The Claudius' cell layer on the basilar membrane was dissected from the basal turn of the cochlea. A voltage-sensitive vibrating probe was used to measure transepithelial short circuit current (Isc ). The baseline Isc of Claudius' cells was measured in the perilymph-like control solution and the change of Isc after application of amiloride (10 μM) or uridine triphosphate (UTP, 100 μM). Results: A negative baseline Isc was observed in the control solution (–12.50 ± 3.95 μA/cm2, n = 8) and the addition of amiloride resulted in a decrease of Isc by 75.8%. The application of UTP, an agonist for P2Y purinergic receptors, led to a partial inhibition of Isc (by 38.2 ± 3.2%, n = 5), and subsequent addition of amiloride abolished the remaining Isc.

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.

BACKGROUND: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.RESULTS: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.CONCLUSIONS: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.

BACKGROUND: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].RESULTS: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+.CONCLUSIONS: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.

Background The low luminal Ca 2+ concentration of mammalian endolymph in the inner ear is required for normal hearing and balance. We recently reported the expression of mRNA for a Ca 2+ -absorptive transport system in primary cultures of semicircular canal duct (SCCD) epithelium. Results 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 Ca 2+ -ATPase (PMCA1, PMCA2, PMCA3, and PMCA4) in native SCCD, cochlear lateral wall (LW) and stria vascularis (SV) of adult rat as well as Ca 2+ channels in neonatal SCCD. All components were expressed except TRPV6 in SV and PMCA2 in SCCD. 1,25-(OH) 2 vitamin D 3 (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. Conclusions These findings demonstrate for the first time the expression of a complete Ca 2+ 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.