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The low Ca 2+ 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 Ca 2+ channels TRPV5 and TRPV6 are expressed in the vestibular system and that TRPV5 expression is stimulated by 1,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ), as also reported in kidney. TRPV5/6 channels are known to be inhibited by extracellular acidic pH. Endolymphatic pH, [Ca 2+ ] and transepithelial potential of the utricle (UP) were measured in Cl -/ exchanger pendrin (SLC26A4) knockout mice in vivo. Slc26a4 -/-mice exhibit reduced pH and UP and increased [Ca 2+ ]. Monolayers of primary cultures of rat semicircular canal duct (SCCD) cells were grown on permeable supports and cellular uptake of 45 Ca 2+ was measured individually from the apical and basolateral sides. Net uptake of 45 Ca 2+ was greater after incubation with 1,25(OH) 2 D 3 . Net 45 Ca 2+ 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 [Ca 2+ ] due to luminal acidification and consequent inhibition of TRPV5/6-mediated Ca 2+ absorption.

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.

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BackgroundThe 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.ResultsWe 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.ConclusionsThese 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.