Pflügers Archiv

, Volume 453, Issue 1, pp 11–22

What’s new in ion transports in the cochlea?

Authors

    • Inserm EMI U-0112Faculté Xavier Bichat
    • Pediatric ENT DepartmentHôpital Necker–Enfants Malades
  • Olivier Sterkers
    • Inserm EMI U-0112Faculté Xavier Bichat
    • ENT DepartmentHôpital Beaujon
  • Evelyne Ferrary
    • Inserm EMI U-0112Faculté Xavier Bichat
    • ENT DepartmentHôpital Beaujon
Invited Review

DOI: 10.1007/s00424-006-0103-4

Cite this article as:
Couloigner, V., Sterkers, O. & Ferrary, E. Pflugers Arch - Eur J Physiol (2006) 453: 11. doi:10.1007/s00424-006-0103-4

Abstract

Recent advances in the field of the physiology of inner ear fluids permitted the characterization of the molecular mechanisms involved in critical processes such as the absorption of K+ through cochlear sensory hair cells (mechanoelectrical transduction) or the secretion of K+ by marginal cells of the stria vascularis. In addition, new pathways for ion circulations were evidenced. Mutations of transporters involved in some of these pathways, especially in K+ recycling through gap junction systems, and in local pH regulation, are among the most frequent etiologies of genetic deafness in humans.

Keywords

Inner ear fluidsPotassiumSodiumAcid-baseEndolymphConnexinHearingDeafness

Introduction

The inner ear encompasses three organs, the cochlea, responsible for hearing; the vestibule, sensitive to gravity and accelerations; and the endolymphatic sac, devoid of sensory function. It consists of an epithelium named the membranous labyrinth, and of several fluid compartments (Fig. 1). The membranous labyrinth is a heterogeneous tight epithelium whose cells can be divided into four cellular populations: sensory cells, secretory cells, other cells such as supporting cells, and cochlear fibrocytes. Recently, the gap junction networks that connect these cells were shown to play a critical role in the homeostasis of inner ear fluids. These fluids are endolymph, a K+-rich fluid that bathes the apical side of the membranous labyrinth, and perilymph, a Na+-rich fluid that bathes its basolateral side. The study of inner ear physiology has gone through three successive steps: transepithelial (for review, see [55]), cellular (for review, see [65]), and, more recently, molecular. Lists of the various types of syndromic and nonsyndromic deafness resulting from mutations of genes encoding inner ear proteins are provided by the following websites: http://www.jax.org/hmr/models.html (mice) and http://webhost.ua.ac.be/hhh/ (humans). The aim of the present review is to integrate molecular data in our understanding of the physiology and pathophysiology of inner ear fluids.
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Fig. 1

Cochlear tissues and fluid compartments. a Cochlear fluids. Endolymph is the fluid that bathes the apical side of the membranous labyrinth. It is K-rich, positively polarized, and is at the base of the cochlea; it is hyperosmotic compared to plasma. It originates from perilymph. The basolateral side of the cochlear membranous labyrinth is surrounded by several NaCl-rich extracellular fluids: perilymph of the scala tympani (ST) under the basilar membrane, perilymph of the scala vestibuli (SV) above Reissner’s membrane, the intrastrial space between the basal and the marginal cells of the stria vascularis, and cortilymph between the basolateral membranes of the organ of Corti cells and the basilar membrane. Tracer diffusion studies showed that there is no diffusion barrier between perilymph of scala tympani, scala vestibuli, spiral ligament, cortilymph, and spiral limbus. Thus, all these fluid compartments can be called perilymphatic spaces. At variance, the intrastrial space constitutes an isolated compartment separated from surrounding fluids by tight junctions between strial marginal and basal cells. In the endolymphatic sac, the luminal fluid has an original composition because it is Na-rich as perilymph, and positively polarized as endolymph. b Cochlear tissues. The cochlear sensory patch, named organ of Corti, lies on the basilar membrane. In the organ of Corti, sensory hair cells are composed of three rows of outer hair cells and one row of inner hair cells. Only inner hair cells are true sensory cells whereas outer hair cells amplify the signal and increase its frequency selectivity. Within the organ of Corti, hair cells are surrounded by several types of supporting cells. Reissner’s membrane is a thin and mobile membrane composed of epithelial and mesothelial layers. Located in the lateral wall of the cochlea, stria vascularis (Stria V) is a multilayered, highly vascularized epithelium. It is composed of marginal cells in contact with endolymph, neural crest-derived intermediate cells, and basal cells. Below the stria vascularis lies the spiral prominence (Sp P), a monolayer epithelium. Stria vascularis, spiral prominence, and outer sulcus cells, the most lateral supporting cells, are bordered laterally by a loose connective tissue named the spiral ligament (Sp Lig). The spiral limbus (Sp Limb) is located between the organ of Corti laterally, the spiral osseous lamina inferiorly, and the tectorial membrane (TM) superiorly. It contains fibrocytes and columns of epithelial cells named interdental cells. The compartment delimited by the cochlear lateral wall, the basilar membrane inferiorly, and Reissner’s membrane superiorly, is named scala media or cochlear duct

Cochlear gap junctions

During the last decade, many studies pointed out the existence and the functional relevance of cochlear gap junction systems. Gap junctions enable the free diffusion of small molecules (<1,000 Da) such as nutrients, second messengers, or ions between interconnected cells. They constitute intercellular protein channels composed of two hemichannels (one for each connected cell) named connexons. Each connexon is formed by the homo- or heterotypic assembly of six proteins named connexins. Cochlear gap junctions were evidenced by the analysis of the cochlear ultrastructure and by connexins immunostaining (see the nature of the connexins involved in the gap junction systems in Table 1). Their functional relevance in cochlear physiology is strongly suggested by their abundance. Thus, in supporting cells of the organ of Corti, they occupy more than 25% of the plasma membrane area.
Table 1

The connexins involved in the cochlear gap junction systems

Connexin

Gene

Location

Deafness

26

GJB2

Supporting cells of the organ of Corti

DFNA3

Basal cell region stria vascularis

DFNB1

Fibrocytes type I and V spiral ligament

 

Spiral limbus

 

30

GJB6

Supporting cells of the organ of Corti

DFNA3

Basal cell region stria vascularis

DFNB1

Fibrocytes type V spiral ligament

 

Spiral limbus

 

31

GJB3

Type II fibrocytes below spiral prominence

DFNA2

43

GJA1

Stria vascularis

Spiral ligament (lateral zone)

 

Outer sulcus cells

 

Spiral limbus

 

Mouse type I fibrocytes

 

45

GJA7

Capillaries (mouse)

The gap junction networks

Two gap junction networks were described: the epithelial cell and the connective tissue gap junction systems (for review, see [23, 53]) (Fig. 2).
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Fig. 2

Putative transcellular routes for cochlear K+ flow. Mechanoelectrical transduction is the transformation of acoustic stimuli into nervous inputs. K+ flow through cochlear hair cells is an essential step of this process, leading to the following succession of events: hair cells depolarization, increase in intracellular Ca++ concentration, release of glutamate within the synaptic space between the hair cell and the auditory nerve afferent fiber, and stimulation of the afferent fiber. K+ can leave the endolymph through nonsensory cells, especially through outer sulcus cells. The probable function of this parasensory pathway is to compensate the variations of the sensory route though hair cells resulting from fluctuations of the acoustic environment. K+ ions present in hair cells of the basolateral compartment probably circulate through the epithelial gap junction system, eventually reaching the perilymphatic spaces of the spiral limbus medially and spiral ligament laterally. K+ ions present in the perilymphatic space of the spiral ligament could circulate through the cochlear lateral wall connective tissue cell gap junction system and eventually reach the intrastrial fluid (ISF). From there, K+ is secreted in endolymph by strial marginal cells via an apical K+ conductance, KCNE1/KCNQ1. The endocochlear potential is generated by the release of K+ within the intrastrial space through strial intermediate cells KCNJ10 K+ channel. In addition, K+ ions present in the perilymphatic space of the spiral limbus may circulate through the spiral limbus connective tissue cell gap junction system and through interdental cells, eventually reaching endolymph (EL). B Basal cells of stria vascularis, IC intermediate cells of stria vascularis, IDC interdental cells, PLT perilymph of the scala tympani, PLV perilymph of the scala vestibuli, S Limb F fibrocytes of the spiral limbus, I to V types I to V spiral ligament fibrocytes, green area epithelial cells gap junction system, orange area connective cells gap junction system, dark blue area intrastrial fluid, black arrows routes for K+ secretion of endolymph from perilymph, white arrows sensory routes for K+ recycling from endolymph to endolymph, and yellow arrows parasensory routes for K+ recycling from endolymph to endolymph [9, 23, 53]

The epithelial cell gap junction system encompasses all the supporting cells of the organ of Corti from outer sulcus cells laterally to interdental cells medially (Fig. 2). In contrast, there is no gap junction between the hair cells and the supporting cells. At its proximal and distal ends, the epithelial cell gap junction system is separated from the adjacent connective tissue cells by a continuous basement membrane. Laterally, this system connects the outer hair cells of the basolateral compartment with the spiral ligament extracellular space, and more specifically with spiral ligament type II fibrocytes that surround the basolateral root processes of outer sulcus cells. Medially, this system connects the inner hair cells of the basolateral compartment with both the spiral limbus extracellular space and the cochlear endolymph through medial interdental cells. The functional link between inner hair cells and the medial part of the epithelial cell gap junction system is supported by the alteration of cells belonging to this system after the damage of inner hair cells by carboplatin. The connective tissue cell gap junction system connects the spiral ligament extracellular space to the intrastrial space (Fig. 2). It also links the perilymph of scalae to the intrastrial space. Medially, the connective tissue cell gap junction system consists of spiral limbus fibrocytes, some of which are close to the scala vestibuli (supralimbal fibrocytes), scala tympani, inner sulcus cells, and interdental cells.

Functional relevance of the cochlear gap junction systems

The role of the connexins in the cochlear physiology was largely studied. In humans, mutations of connexins 26 and 30 are by far the most frequent etiologies of nonsyndromic genetic deafness (Table 1) [49].

In gerbils, the in vivo perilymphatic infusion of proadifen (SKF-525A), a gap junction uncoupler, reduced the distortion product otoacoustic emissions and increased the compound action potential thresholds [54]. In parallel, a vacuolization of types II and V spiral ligament fibrocytes was observed.

In mice, C×26 (10,27) and C×30 (60) loss-of-function mutations lead to sensorineural hearing loss and to degeneration of hair cells and supporting cells, showing the critical role of the epithelial cell gap junction system in the survival of hair cells. The role of the lateral connective cell gap junction system is supported by the phenotype observed in Brn-4 null mutant mice. BRN-4 gene encodes Brn-4, a transcription factor belonging to the POU family. In humans, its mutations induce DFN3, an X-linked nonsyndromic deafness. In the mouse cochlea, Brn-4 mRNA is specifically expressed in type I, II, III, and V spiral ligament fibrocytes [40]. Brn-4 null mutant mice are profoundly deaf. The only morphological anomaly observed in their cochlea is a decrease in the cellular volume, mitochondria content, and cytoplasmic extensions of spiral ligament fibrocytes.

Cochlear gap junction systems are probably involved in ionic transports (see below) but also in the intercellular diffusion of larger molecules such as second messengers. Thus, the in vitro analysis of gap junctions containing mutated C×26 (five mutations involved in human deafness) showed that 60% (3/5) of the studied mutations were associated with a normal junctional conductance [4, 45, 62]. In cells expressing the V84L mutation, the functional anomaly that was detected was a reduced permeability of gap junctions to Ins(1,4,5)P3 [4]. The link between the decreased permeability of V84L gap junctions to Ins(1,4,5)P3 and deafness remains to be determined. In vitro experiments showed that the intercellular diffusion of Ins(1,4,5)P3 triggers the propagation of Ca++ waves, which activate the endolymphatic secretion of Cl through supporting cells of the apical Cl conductances [4].

Finally, it should be pointed out that the fibrocytes belonging to the gap junction systems have other functions other than the transcellular diffusion of small molecules, such as the buffering of mechanical constraints generated by sound vibrations or the synthesis of extracellular proteins like cochlin.

Cochlear circulation of potassium

Cochlear K+ flows are both transcellular and paracellular. Some transcellular flows were well established, such as the mechanoelectrical transduction, i.e., the transformation of acoustic stimuli into nervous inputs performed by sensory hair cells, and the secretion of K+ by stria vascularis. More recently, additional pathways involving other cochlear cell populations were characterized (Fig. 2).

K+ absorption in sensory hair cells

The fact that K+ provides the major charge carrier for the cochlear mechanoelectrical transduction has two major advantages: (1) Because K+ is by far the most abundant ion in the cytosol, an influx of K+ ions into the sensory cells causes the least change in the cytosolic concentration compared to any other ion; (2) K+ flows through hair cell of the apical and basolateral membranes are energetically inexpensive because both flows occur down an electrochemical gradient.

K+ crosses hair cells of the apical membrane through nonselective cation channels. The molecular nature of these channels remains to be determined. TRPA1, a member of the TRP family of ion channel proteins, is a good candidate for this function [43]. K+ is then released through basolateral K+ channels, probably KCNQ4, in outer hair cells [48]. Indeed, chronic intracochlear perfusion of linopirdine, a pharmacological inhibitor of KCNQ channels, elicits a degeneration of outer hair cells [44]. KCNQ4 mutation is responsible for DFNA2, an autosomal dominant genetic deafness. The role of BK, another K+ channel located in hair cells of the basolateral membrane, remains to be determined. The deafness observed in BK α-subunit-deficient mice is apparently due to the disappearance of KCNQ4 in outer hair cells [48].

K+ absorption through outer sulcus cells

In the gerbil, an apical-to-basal transepithelial cation current through outer sulcus cells was evidenced [9]. Passive K+ absorption through outer sulcus cells is mediated by an apical P2X2 ligand-gated nonselective cation channel and a large basolateral K+ conductance. Absorption of K+ from endolymph to spiral ligament constitutes a parasensory flow of K+ out of endolymph. The function of this parasensory pathway is probably to compensate the variations of the sensory route though hair cells resulting from fluctuations of the acoustic environment. In addition, some of the K+ ions that reach outer sulcus cells probably come from outer hair cells of the basolateral compartment through the epithelial cell gap junction system. The vicinity between outer sulcus cells root processes and spiral ligament type II fibrocytes suggests that part of the K+ released basolaterally by outer sulcus cells is taken up by the lateral connective tissue cell gap junction system.

Gap junction systems and cochlear K+ flows

One of the putative functions of the cochlear gap junction systems is to favor rapid intracellular K+ diffusion through the organ of Corti, the spiral limbus, and the spiral ligament.

K+ flow through the epithelial cell gap junction system

Once K+ ions have crossed hair cells during mechanoelectrical transduction, they are probably rapidly cleared out of their basolateral compartment to avoid local accumulation of K+ with subsequent hair cells degeneration. The degeneration of hair cells observed in C×26 and C×30 null mutant mice is compatible with this hypothesis [10, 27].

Much of the K+ discharged in the basolateral compartment of hair cells is probably taken up via KCC4, an electroneutral K-Cl cotransporter located in two types of supporting cells, Deiters’ cells around outer hair cells and phalangeal cells around inner hair cells [7]. KCC4-deficient mice develop a sensorineural deafness associated with a progressive degeneration of the organ of Corti [7]. The first cells that disappear are outer hair cells. The fact that the cellular degeneration begins after postnatal day 14, i.e., after the onset of mechanoelectrical transduction, supports the hypothesis that this degeneration results from a local accumulation of K+. Spicer and Schulte [53] postulate that the K+ ions that diffuse through the epithelial cell gap junction system are partly recycled into endolymph along lateral and medial recycling pathways involving the connective tissue cell gap junction system and interdental cells. In particular, there may be a direct link between the epithelial cell gap junction system and endolymph through the lateral columns of interdental cells, which are connected with inner sulcus cells on one hand and which face endolymph on the other hand [53]. These K+ recycling pathways are not essential to the endolymphatic secretion of K+ because the dysfunction of the epithelial cell gap junction system in KCC4 null mutant mice [7] or in mice with a targeted ablation of C×26 in the epithelial cell gap junction system [10] does not alter the endocochlear potential nor the endolymphatic K+ concentration and volume.

K+ flows through the connective tissue gap junction system

One of the probable functions of the connective tissue gap junction system is to facilitate K+ flow from the perilymphatic spaces to cells involved in the endolymphatic secretion of K+, strial marginal cells laterally, and maybe interdental cells medially.

Several K+ transporters were localized in fibrocytes located at the interface between the perilymphatic spaces and the connective tissue cell gap junction system. Their role is probably to take up K+ from perilymphatic compartments into gap junction networks by similar mechanisms as the ones used by strial marginal cells to take up K+ from intrastrial space (see below). Indeed, as in the basolateral plasmalemma of marginal cells, Na+/K+-ATPase (α1β1 and α2β1 isoforms), NKCC1 or SLC12A2, the “secretory” isoform of the Na+/K+/2Cl cotransporter, and the CLC-KB Cl channel were detected by immunohistochemistry in spiral limbus fibrocytes and in types II, IV, and V spiral ligament fibrocytes. In support of an intense transport activity, type II, IV, and V spiral ligament fibrocytes have numerous surface projections and intracellular mitochondria, and the intensity of their Na+/K+-ATPase immunostaining is similar to the one observed in strial marginal cells. An additional K+ transporter found in the spiral limbus and in the spiral ligament type II, IV, and V fibrocytes is Kir5.1, an inward rectifying K+ channel [19]. It is interesting to note that there is a temporal correlation between the developmental expression of Kir5.1 subunits in spiral ligament fibrocytes and the phase of rapid elevation of endocochlear potential.

The deafness observed in case of loss of expression of NKCC1 [13] and of Barttin, a regulating subunit associated with CLC-K Cl channels [6], might partly result from the functional role of these transporters in cochlear gap junction systems in addition to their role in strial marginal cells.

Three voltage-gated K+ channels were detected along the cochlear gap junction systems:
  • Ether-a-go-go K+ channel mRNA, localized by in situ hybridization in rat-supporting cells, mainly Deiters cells, and in spiral ligament type I, III, IV, and V fibrocytes [30];

  • Kv3.1b, detected by immunohistochemistry in type I, II, and V spiral ligament fibrocytes, spiral limbus and supralimbal fibrocytes, and interdental cells [52];

  • BK, a voltage- and Ca++-dependent big-conductance K+ channel, detected in cultured gerbil type I fibrocytes [51]. In the same cells, the cytosolic concentration of free Ca++, which is critical to the activity of BK channels, is regulated by two calcium transporters, the alpha1C isoform of the L-type Ca++ channel encoded by the Cav1.2 gene, and the intracellular SERCA Ca++-ATPase [36]. In BK channel α and β1 subunits null mice, there was no obvious morphologic abnormality of the cochlear lateral wall [48].

These three channels might regulate the gap junction conductance, which was shown to be sensitive to membrane voltage, by controlling the intracellular potentials [30, 52].

K+ release from the spiral ligament gap junction network into the intrastrial space is responsible for the generation of the endocochlear potential

Morphologic studies strongly suggest that after diffusing through the spiral ligament gap junction network, K+ is released in the intrastrial space by the basal or by the upper type of strial intermediate cells. At least part of this K+ release occurs through KCNJ10 (Kir4.1), a K+ channel specifically localized in strial intermediate cells and whose developmental expression parallel the formation of the endocochlear potential [1]. The K+ flow through KCNJ10 generates the endocochlear potential. Indeed, measurement of the potential difference between the outer surface of the cochlear lateral wall and various areas within this wall showed that the endocochlear potential is generated across the basal cell barrier rather than across the marginal cell barrier. Furthermore, a comparison between the drug sensitivity profiles of the endocochlear potential and of the currents through KCNJ10 K+ channels supports the hypothesis that KCNJ10 generates the endocochlear potential. Finally, there is no endocochlear potential in mutant mice (Wv/Wv) lacking strial intermediate cells, and in KCNJ10 knockout mice [39]. In the latter mice, the cochlear endolymphatic volume and K+ concentration are decreased, strongly supporting the hypothesis that K+ flow from perilymph to the intrastrial space mainly occurs through the spiral ligament gap junction network. The residual secretion of endolymph observed in these mice might occur through additional strial K+ conductances such as the maxi-K K+ channel present in strial basal cells [59], or medially through interdental cells. In C×30 and Brn-4 null mutant mice, and after perilymphatic treatment with the gap junction uncoupler proadifen, the endocochlear potential is substantially reduced [40, 54, 60], confirming the participation of the connective tissue cell gap junction system in K+ transport toward the intrastrial space. However, in none of these models were the morphology of the stria vascularis and the endolymphatic volume and concentration modified. This may be due to the fact that K+ diffusion through cochlear gap junction networks is only partially inhibited in these experimental models, or that other K+ pathways compensate the inhibition of gap junctions. C×26 seems to play a minor role in the K+ flow through the connective tissue cell gap junction system because the transgenic expression of a dominant negative C×26 does not alter the endocochlear potential, nor the endolymphatic secretion of K+ [27].

Endolymphatic K+ secretion by strial marginal cells

Strial marginal cells take up K+ across their basolateral membrane via a Na+/K+/2Cl cotransporter, SLC12A2 (previously called BSC2 or NKCC1), and Na+/K+-ATPase. They secrete K+ across the apical membrane via a K+ channel, KCNQ1/KCNE1 (formerly called KvLQT1/IsK channel) (for review, see [65]).

Basolateral K+ transporters

The combination of basolateral Na+/K+-ATPase and Na+/K+/2Cl cotransporter is very energy-efficient because theoretically, five K+ ions are taken up per one ATP hydrolyzed: Hydrolysis of one ATP molecule enables the Na+/K+-ATPase to transport two K+ ions within the marginal cell and to extrude three Na+ ions in the intrastrial space. These three Na+ ions power three cycles of the Na+/K+/2Cl cotransporter, leading to the additional entry of three K+ ions within the marginal cell.

The Na+/K+-ATPase subunit isoforms expressed in stria vascularis are α1 and β2; β2 is not expressed in any other cochlear tissue.

SLC12A2 is the Na+/K+/2Cl cotransporter involved in K+ secretion in the basolateral membrane of strial marginal cells. In mice lacking SLC12A2, the cochlear endolymphatic space is collapsed due to the inability of strial marginal cells to secrete K+ [13]. In humans, no deafness linked with a mutation of the gene encoding SCL12A2 was described yet. Cl entering the marginal cells through SLC12A2 are recycled back to the intrastrial space through basolateral CLC-KA and CKC-KB Cl channels [2]. ClC-KA or ClC-KB gene mutations do not induce deafness, the persistence of one CLC-K channel probably compensating the loss of the other one. At variance, mutations of the gene encoding for Barttin, a small regulating subunit associated with both channels, induce type IV Bartter syndrome, a disease associated with a sensorineural hearing loss [6].

Apical KCNQ1/KCNE1 K+ channel

K+ crosses strial marginal cells of the apical membrane through the KCNQ1/KCNE1 K+ channel. KCNQ1 and KCNE1 have distinct functions within the K+ channel: KCNQ1 is the K+ conductance, whereas KCNE1 may be necessary for tracking of KCNQ1 to the apical membrane. Besides the inner ear, KCNQ1/KCNE1 is also expressed in the heart where it plays a major role in the repolarization phase of the cardiac action potential. In mice lacking either KCNE1 [61] or KCNQ1 [47], the endolymphatic spaces develop normally until about postnatal day 3, a date that in mice, corresponds to the onset of K+ secretion. Later, the cochlear endolymphatic space appears to be collapsed due to the inability of strial marginal cells to secrete K+ and due to unimpeded reabsorptive processes that may have a similar onset in development [61]. In humans, mutations of KCNQ1/KCNE1 are responsible for two syndromes:
  • The Romano–Ward syndrome, an autosomal dominant disease, which consists of an isolated impairment of cardiac ventricular repolarization, named long-QT syndrome. The main clinical manifestations of this syndrome are arrhythmias, syncope, and sudden death; and

  • The Jervell and Lange-Nielsen syndrome, an autosomal recessive disease, which associates long-QT syndrome and profound sensorineural deafness. In patients with Jervell and Lange-Nielsen syndrome, the endolymphatic space is collapsed, as observed in mice lacking either KCNQ1 or KCNE1.

K+ flow through Reissner’s membrane

Reissner’s membrane epithelial layer, which lines endolymph and whose cells are linked with tight junctions, probably regulates ion transports through the membrane. The main pathway for K+ flow across it associates an apical stretch-activated nonselective cation channel and a basolateral Na+/K+-ATPase [67]. The apical stretch-activated channel is activated by pressures exceeding 15 mmHg [67]. Reissner’s membrane gets distended by similar pressure increases. Thus, in case of pathological increase in the volume of cochlear endolymph, named endolymphatic hydrops, the apical stretch-activated channels may be activated and may reduce the volume of endolymph by enabling a K+ leak out of endolymph.

Role of the tight junctions

All the epithelial cells lining cochlear endolymph are linked with tight junctions that limit the intercellular diffusion of molecules. In addition, in the stria vascularis, basal strial cells are also endowed with tight junctions so that the intrastrial space is electrically isolated. The main tight junction proteins are named claudins. They are thought to create charge-selective channels that regulate transepithelial paracellular ionic flows. In claudin-11-null mice, which lack the strial basal cell tight junctions, the endocochlear potential is decreased below 30 mV and there is a 50-dB hearing loss [17, 24]. In these mice, the endolymphatic K+ concentration and the cochlear morphology are normal, and the changes in the expression of genes associated with K+ homeostasis are, at most, minor. These results are in accordance with the mechanisms of generation of the endocochlear potential described above.

In humans, mutations in the gene encoding tight junction claudin-14 cause autosomal recessive deafness DFNB29. Claudin-14 is expressed by inner and outer hair cells and by supporting cells [5]. Claudin-14 null mutant mice have a 50- to 60-dB hearing loss [5]. Their hair cells, predominantly outer hair cells, degenerate, whereas their endocochlear potential is normal. The tight junction strands between supporting cells and between outer hair cells and supporting cells appear normal, probably because these tight junctions contain other claudins in addition to claudin-14. Organ of Corti explants of Cldn14−/− mice survived as long as those of Cldn14+/+ mice, suggesting that hair cell death in Cldn14+/+ mice is not triggered by a signal intrinsic to the cell, but by extracellular factors. Finally, transfection of a renal cell line named MDCKII with claudin-4, -14, and -15 show that claudin-14 specifically decreases the paracellular permeability to both K+ and Na+. These data support the hypothesis that claudin-14 is responsible for the restriction of cations diffusion through the tight junction network of the organ of Corti. The degeneration of hair cells in Cldn14−/− mice probably results from the accumulation of K+ within hair cells of the basolateral compartment. This mechanism was also postulated to be responsible for hair cells degeneration in KCC4 null mutant mice, in mutant mice that do not express C×26 in the epithelial cells gap junction system, and in animals in which the KCNQ4 K+ channel located within the basolateral membrane of outer hair cells was inhibited by chronic intracochlear perfusion of linopirdine.

Cochlear flows of sodium

Na+ absorption is necessary to maintain the low concentration of Na+ in endolymph. The Na+ transporters, epithelial sodium channel (ENaC) Na+ channel, nonselective cation channels, and Na+/H+ exchanger, were localized by in situ hybridization and immunohistochemistry in several types of endolymph lining cells. Nevertheless, electrophysiological studies suggest that Reissner’s membrane and outer sulcus cells are the two major sites for Na+ absorption.

Na+ absorption through Reissner’s membrane

The majority of the short circuit current produced by Reissner’s membrane isolated from gerbil was inhibited by amiloride analogs (benzamil>amiloride>>ethylisopropylamiloride), consistent with Na+ absorption through the ENaC epithelial sodium channel in the apical cell membrane [33]. The presence of ENaC in Reissner’s membrane is also supported by the study of the distribution of ENaC by in situ hybridization [12] and immunohistochemistry [41]. The electrochemical driving force that drives Na+ entrance through the apical ENaC channel is probably about 90 mV [33]. In isolated Reissner’s membrane, the short circuit current was also inhibited by an inhibitor of Na+/K+-ATPase, ouabain, and by the K+ channel blockers Ba++, 4-aminopyridine, and quinine, but not tetraethylammonium nor glibenclamide; this is consistent with the presence of a voltage-activated K+ channel [33]. Thus, the Na+ ions that enter the cell through apical ENaC are probably pumped out into the perilymphatic compartment by basolateral Na+/K+-ATPase. The K+ entering the cell through this pump is recycled toward the basolateral compartment by a K+ channel.

Na+ absorption through outer sulcus cells

As shown by the study of the transepithelial short circuit current of isolated tissue, outer sulcus cells probably represent another area of Na+ absorption out of endolymph [34]. Na+ would cross these cells through an apical P2X2 ligand-gated nonselective cation channel and basolateral Na+/K+-ATPase. The same apical channels are involved in parasensory K+ recycling through the cochlear lateral walls. The Na+ current measured in vitro through outer hair cells is 25% smaller than the one measured through Reissner’s membrane under the same conditions (high [K+] and low [Na+]) [33]. Moreover, the surface of Reissner’s membrane is much larger than the overall surface of outer sulcus cells, so that in vivo, the Na+ current through Reissner’s membrane is probably substantially larger than the one through outer sulcus cells.

Na+ flow toward the intrastrial space

The presence of a high concentration of Na+ in the intrastrial space is essential to the process of K+ secretion through strial marginal cells. The mechanisms by which Na+ ions reach the intrastrial space remain unclear. The perilymphatic administration of bumetanide, an inhibitor of the Na+/K+/2Cl cotransporter; of ouabain, an inhibitor of basolateral Na+/K+-ATPase; and of Na+-poor solutions show that the concentration of Na+ within the intrastrial space is:
  • Increased by the inhibition of the Na+/K+/2Cl cotransporter. This inhibition probably results from the inhibition of Na+ uptake from the intrastrial space by the marginal cells of the basolateral Na+/K+/2Cl cotransporter. The diffusion of bumetanide to marginal cells through the barrier of strial basal cells layer is possible because this inhibitor is a lipophilic agent; and

  • Decreased by the inhibition of Na+/K+-ATPase and by the removal of Na+ from perilymph [3, 20]. The hypothesis that best fits this observation is that Na+ reaches the intrastrial space through the spiral ligament. Na+ could enter spiral ligament fibrocytes through the ENaC Na+ conductances that are extensively distributed in the spiral ligament. Na+ could then diffuse through the spiral ligament gap junction network and could then be released in the intrastrial space via strial intermediate cells Na+/K+-ATPase.

The contribution of Na+ absorption to the endocochlear potential

The Na+ absorption out of endolymph under basal conditions would be expected to contribute only a small negative component to the endocochlear potential because the resting absorptive flux of Na+ from cochlear endolymph was estimated to represent only 1% of K+ secretory flux [26]. Consistent with this notion, endolymphatic perfusion of amiloride in K+-rich solution had no significant impact on the endocochlear potential [12].

Possible involvement of epithelial sodium channel in genetic deafness

No deafness was described in patients presenting with mutations of ENaC subunits. TMPRSS3, a transmembrane serine protease, is present in several cochlear tissues (stria vascularis, supporting cells of the organ of Corti, and spiral ganglion) [18]. Mutations of TMPRSS3 are responsible for the nonsyndromic autosomal recessive deafness DFNA8/10. In the Xenopus oocyte, TMPRSS3 was shown to activate ENaC after undergoing a process of activation by autocatalytic proteolysis [18]. Thus, it is possible that inner ear ENaC is a substrate of TMPRSS3 and that a dysfunction of ENaC is involved in the pathogenesis of DFNA8/10.

Cochlear acid-base transports

Due to the positivity of the endocochlear potential, the maintenance of a physiological pH of 7.4 in endolymph requires an active secretion of H+ within endolymph. A good candidate for this secretion is the apical vacuolar H+-ATPase localized in different parts of the cochlear membranous labyrinth. An isoform of the B subunit of the v-H+-ATPase, encoded by the ATP6V1B1 gene, is specifically expressed in the inner ear (cochlear spiral limbus and endolymphatic sac) and in the renal distal tubule [22]. In humans, mutations of ATP6V1B1 and of ATP6V0A4, other genes encoding for a v-H+-ATPase subunit isoform, induce an autosomal recessive syndrome associating renal distal tubular acidosis and deafness.

Carbonic anhydrase and HCO3 exchangers are also present in several tissues facing endolymph. In humans, mutations of SLC26A4, a gene that encodes for pendrin, a putative HCO3 exchanger, induce an autosomal recessive deafness, sometimes (Pendred syndrome) but not always (DFNB4) associated with a thyroid dysfunction. In mice, pendrin is present within the cochlear lateral wall (outer sulcus cells, root cells, spiral prominence cells, and stria vascularis) [66]. In SLC26A4 null mutant mice, the endocochlear potential is absent, the endolymphatic K+ concentration is normal, the thickness of stria vascularis and of the spiral ligament area normally occupied by type I and II fibrocytes is reduced, and the volume of cochlear endolymph is increased as shown by the bulging of Reissner’s membrane [66]. The absence of endocochlear potential in these mice is probably due to the loss of expression of the KCNJ10, a K+ channel located in stria vascularis intermediate cells and involved in the generation of the endocochlear potential. The KCNJ10 protein was absent in spite of the presence of mRNA encoding for this protein. The mechanisms leading to the loss of KCNJ10 and to the increase in the volume of cochlear endolymph remain to be determined.

Cochlear water flows

Cochlear water flows are still poorly understood. The main conceptual difficulty is the coexistence of cochlear endolymph hyperosmolarity compared to perilymph and plasma, in spite of the fact that the perilymph–endolymph barrier is 130 times more permeable to water than to K+ (for review, see [55]).

In absorptive and secretory epithelial such as the kidney, transepithelial water flows depend on the insertion of water channels named aquaporins (AQP) within the plasmalemma. Several types of AQP were detected within the cochlea (Table 2). The most abundant ones are AQP-1 and AQP-4, and their expression is dramatically upregulated during development [21]. No case of human deafness due to mutation of a gene encoding for an AQP was reported yet. This may result from the functional redundancy of the several AQP present in the inner tissues. The only mice with a genetically modified expression of AQP that were shown to be deaf are AQP-4 null mutant mice [35]. The deafness is due to an inner ear dysfunction (endocochlear hearing loss) and concerns all frequencies. The cochlear histology is normal. AQP-4 immunostaining suggests that this channel might play a role in the regulation of intracellular osmolarity in supporting cells of the organ of Corti. Indeed, these cells are submitted to important osmotic stresses resulting from K+ recycling from hair cells of the basolateral compartment to stria vascularis.
Table 2

The distribution of AQP within the mammalian cochlea

Aquaporin

Location

Technique

AQP-1

Spiral ligament

Immunolocalization

Spiral limbus fibrocytes

Mesenchymal cells below the basilar membrane and in the perilymphatic space

Stria vascularis

Organ of Corti

AQP-2

Reissner’s membrane

Immunolocalization

Organ of Corti

Inner and outer sulcus cells

Spiral limbus

AQP-3

Spiral ligament and spiral limbus fibrocytes

Immunolocalization

Organ of Corti

AQP-4

Organ of Corti

Immunolocalization In situ hybridization

Inner sulcus cells

Spiral ligament

AQP-5

Apical cochlear lateral wall

Immunolocalization

Outer sulcus

AQP-6

RT-PCR

AQP-7

Reissner’s membrane

Immunolocalization

Stria vascularis

Supporting cells (Deiters’, Hensen’s, inner phalangeal, and border cells)

AQP-9

Reissner’s membrane

Immunolocalization

Spiral limbus interdental cells

The heterogeneity in the reported distribution of cochlear AQP from one study to the other must be pointed out. This heterogeneity might result from interspecies variations or from differences in the sensitivity and specificity of the techniques used to detect AQP

RT-PCR Reverse transcriptase polymerase chain reaction

Hormonal regulation of cochlear hydroelectrolytic transports

Several endocrine and paracrine systems are involved in endolymph homeostasis.

ATP and acoustic trauma

In the guinea pig, a constitutive level of ATP was reported in both endolymph and perilymph. During acoustic trauma, the concentration of endolymphatic ATP increases [46]. The subsequent stimulation of purinergic receptors in endolymph lining epithelial cells seems to trigger a process that protects the cochlea by lowering the endocochlear potential with subsequent reduction of mechanoelectrical transduction through hair cells. The decrease in the endocochlear potential results from three ATP-induced phenomena:
  • Downregulation of the expression of marginal cells KCNQ1/KCNE1 K+ channel via the activation of apical P2U receptors with protein kinase C as a second messenger [38];

  • K+ flow out of endolymph after increase in expression and activation of ATP-gated ion channels (P2X receptors) present in hair cells, supporting cells from inner sulcus to outer sulcus cells, and some interdental cells [9, 63]; and

  • Endolymphatic secretion of Cl ions through Hensen’s cells of the apical Cl conductances [29].

In guinea pigs submitted to acoustic trauma, the perilymphatic administration of ATP accelerates the restoration of normal hearing [56]. The noise-induced increase in endolymphatic ATP concentration is limited by concomitant upregulation of ectonucleotidases (NTPDases), which degrade ATP.

Other systems, such as the TRPV4 channel, which is expressed in mice hair cells, spiral ganglion cells, and stria vascularis, are probably involved in the protection of the inner ear against acoustic trauma [57].

The arginine vasopressin–aquaporin-2 system

Decrease in blood volume or plasmatic hyperosmolarity elicit an increase in antidiuretic hormone (arginine vasopressin, AVP) plasmatic concentration with subsequent stimulation of renal-collecting duct principal cells AVP V2 receptors. The stimulation of AVP V2 receptors induces an increase in intracellular concentration of the second messenger cyclic AMP (cAMP), which stimulates both the expression of mRNA encoding for AQP-2 and the transfer of AQP-2 proteins from a pool of intracellular vesicles to the apical membrane. The functional outcome of this cascade is an increase in water absorption through the collecting duct with subsequent decrease in diuresis.

In the rat, the administration of AVP upregulated the expression of mRNA encoding for AQP-2, both in the cochlea and the endolymphatic sac [25, 50].

In the guinea pig, the systemic administration of AVP decreased the endocochlear potential, an effect that was reversed by V2 receptor but not by V1 receptor antagonists [42], and elicited the development of an endolymphatic hydrops [28]. In the same species, the injection of cholera toxin, a compound that increases the intracellular concentration of cAMP, AVP V2 receptors second messenger also induced an endolymphatic hydrops [37]. When endolymphatic sac was experimentally obliterated, the administration of specific antagonists of V2 receptors prevented the development of endolymphatic hydrops [58].

In human pathology, no clear relationship was found between AVP–AQP-2 system and Menière’s disease, as syndrome associated with endolymphatic hydrops. No mutation of the gene encoding for AQP-2 was found in patients suffering from Menière’s disease. The results of the dosage of plasmatic AVP concentration in patients suffering from Menière’s disease are contradictory. However, two twins suffering from diabetes insipidus had a Menière’s syndrome and an increased plasmatic AVP concentration [11]. Finally, the value of the plasmatic AVP concentration in Menière’s disease remains under debate. If increased, it might be the consequence rather than the cause of Menière’s symptoms. Indeed, vestibular caloric or electric stimulations were shown to increase plasmatic AVP concentration.

Adrenergic and muscarinic receptors

In the gerbil cochlea, in vitro studies strongly suggest that strial marginal cells K+ secretion is upregulated by β1-adrenergic receptors and downregulated via muscarinic M3- and M4-receptors, cAMP being the second messenger [64].

Steroid hormones

Glucocorticoid, mineralocorticoid, and estrogen receptors were detected in rat, mouse, and human inner ear tissues involved in hydroelectrolytic transports. 11-β-Hydroxysteroid dehydrogenase, an enzyme that is necessary for the specific activation of mineralocorticoid receptors by aldosterone, was localized in the rat spiral ligament.

Functional effects of mineralocorticoids

In null mutant mice that do not express the mineralocorticoid receptor, there is no alteration in the expression of cochlear Na+/K+-ATPase [15]. In guinea pigs, aldosterone alone had no visible effect on the volume of endolymph, but it considerably increased the frequency of endolymphatic hydrops induced by mild experimental lesions of the endolymphatic sac. From this observation, the authors postulated that endolymphatic hydrops might result from the conjunction of an increased secretion of endolymph and a decreased absorption of this fluid by the endolymphatic sac [14].

Functional effects of glucocorticoids

In the rat, the intratympanic injection of glucocorticoids upregulated the expression of AQP-1 and AQP-3 in both the cochlea and the endolymphatic sac [16]. In isolated stria vascularis of gerbils, glucocorticoids increased and mineralocorticoids decreased the transepithelial short-circuit current [43]. These effects were acute, thus, are probably nongenomic [32].

Functional effects of estrogens

In a gerbil preparation of stria vascularis, 17 β-estradiol inhibited the K+ secretion via the KvLQT1 channel in a dose-dependant manner [31]. This effect was acute, and tamoxifen, a competitive inhibitor of intracellular estrogen receptor, did not change the inhibitory effect of 17 β-estradiol. Thus, the effect of this hormone probably resulted from nongenomic mechanisms. In the same study, progesterone induced an acute, small, and transient increase in K+ secretion. The authors state that the hearing impairment observed in case of increase in the plasmatic concentration of 17 β-estradiol, such as hyperfolliculinism, pregnancy, and intake of contraceptive pills, may be, in part, caused by nongenomic inhibition of strial K+ secretion.

Atrial natriuretic peptide

All three atrial natriuretic peptide (ANP) receptors (ANP-A, ANP-B, and ANP-C) were detected in the inner ear of several mammalian species [8]. In the rat, the expression of mRNA encoding the three ANP receptors was upregulated by the administration of ANP in inner ear fluids [8]. This upregulation was reversed by the concomitant administration of anti-ANP antibodies.

Conclusions

The recent advances in the molecular characterization of cochlear hydroelectrolytic transport systems involved in inner fluid physiology has enabled us to better understand the pathophysiological mechanisms underlying several inner ear pathologies, especially in the fields of genetic deafness and Menière’s disease, a cochleovestibular syndrome resulting from an increase in the volume of endolymph. In the future, this knowledge might open the way to the development of new treatments of hearing loss related to inner ear fluids homeostasis disorders.

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© Springer-Verlag 2006