No abstract

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.

The semicircular canal duct (SCCD) epithelium is a vestibular epithelial domain that was recently shown to actively contribute to endolymph homeostasis by Cl(-) secretion under control of beta(2)-adrenergic stimulation. By analogy to other Cl(-) secretory epithelia, we hypothesized that SCCD also provides an active absorptive pathway for Na(+) under corticosteroid control. Measurements of short-circuit current (I(sc)) demonstrated stimulation (7-24 h) by the glucocorticoids hydrocortisone (EC(50) 13 nM), corticosterone (33 nM), prednisolone (70 nM), and dexamethasone (13 nM) over physiologically and therapeutically relevant concentrations and its block by amiloride (IC(50) 470 nM) and benzamil (57 nM), inhibitors of the epithelial sodium channel (ENaC). I(sc) was also partially inhibited by basolateral ouabain and Ba(2+), indicating the participation of Na(+)-K(+)-ATPase and a K(+) channel in Na(+) transport. By contrast, aldosterone stimulated I(sc) only at unphysiologically high concentrations (EC(50) 102 nM). The action of all steroids was blocked by mifepristone (RU-486; K(d) approximately 0.3 nM) but not by spironolactone (K(d) approximately 0.7 microM). Expression of mRNA for the alpha-, beta-, and gamma-subunits of ENaC was demonstrated in the presence and absence of glucocorticoids. These findings are the first to identify SCCD in the vestibular labyrinth as a site of physiologically significant ENaC-mediated Na(+) absorption and osmotically coupled water flux. They further demonstrate regulation of Na(+) transport by natural and therapeutic glucocorticoids. The results provide for the first time an understanding of the therapeutic benefit of glucocorticoids in the treatment of Meniere's disease, a condition that is associated with increased luminal fluid volume.

, which is important for transduction processes. We have recently shown that glucocorticoid receptors (GR) stimulate absorption of Na ϩ by semicircular canal duct (SCCD) epithelia. In the present study, we sought to determine the presence of genes involved in the control of the amiloride-sensitive Na ϩ transport pathway in rat SCCD epithelia and whether their level of expression was regulated by glucocorticoids using quantitative real-time RT-PCR. Transcripts were present for ␣-, ␤-, and ␥-subunits of the epithelial sodium channel (ENaC); the ␣1-, ␣3-, ␤1-, and ␤3-isoforms of Na ϩ -K ϩ -ATPase; inwardly rectifying potassium channels [IC50 of short circuit current (Isc) for Ba 2ϩ : 210 M] Kir2

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. Slc26a4 Δ/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 Slc26a4 Δ/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.