RESUMEN
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 Ca(2+) channels by confocal immunohistochemistry. Cochlear pH and Ca(2+) 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 HCO(3)(-) 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 Ca(2+) reabsorption from endolymph via the acid-sensitive epithelial Ca(2+) channels Trpv5 and Trpv6. Hence the endolymphatic Ca(2+) 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.
Asunto(s)
Ácidos/metabolismo , Proteínas de Transporte de Anión/genética , Bicarbonatos/metabolismo , Calcio/metabolismo , Cóclea/metabolismo , Sordera/genética , Conducto Endolinfático/metabolismo , Mutación , Absorción , Animales , Proteínas de Transporte de Anión/deficiencia , Proteínas de Transporte de Anión/metabolismo , Cóclea/fisiopatología , Sordera/complicaciones , Sordera/fisiopatología , Modelos Animales de Enfermedad , Electrofisiología , Conducto Endolinfático/patología , Gerbillinae , Bocio/complicaciones , Células Ciliadas Auditivas , Concentración de Iones de Hidrógeno , Ratones , Degeneración Nerviosa/genética , Concentración Osmolar , Canales de Potasio de Rectificación Interna/deficiencia , Transportadores de Sulfato , SíndromeRESUMEN
In sea urchin embryos, blastula formation occurs between the seventh and tenth cleavage and is associated with changes in the permeability properties of the epithelium although the structures responsible for mediating these changes are not known. Tight junctions regulate the barrier to paracellular permeability in chordate epithelia; however, the sea urchin blastula epithelium lacks tight junctions and instead possesses septate junctions. Septate junctions are unique to non-chordate invertebrate cell layers and have a characteristic ladder-like appearance whereby adjacent cells are connected by septa. To determine the function of septate junctions in sea urchin embryos, the permeability characteristics of the embryonic sea urchin epithelia were assessed. First, the developmental stage at which a barrier to paracellular permeability arises was examined and found to be in place after the eighth cleavage division. The mature blastula epithelium is impermeable to macromolecules; however, brief depletion of divalent cations renders the epithelium permeable. The ability of the blastula epithelium to recover from depletion of divalent cations and re-establish a barrier to paracellular permeability using fluorescently labelled lectins was also examined. Finally, septate junction structure was examined in embryos in which the permeability status of the epithelium was known. The results provide evidence that septate junctions mediate the barrier to paracellular permeability in sea urchin embryos.
Asunto(s)
Embrión no Mamífero/metabolismo , Desarrollo Embrionario/fisiología , Epitelio/metabolismo , Uniones Intercelulares/metabolismo , Lytechinus/metabolismo , Erizos de Mar/metabolismo , Strongylocentrotus purpuratus/metabolismo , Animales , Transporte Biológico , Embrión no Mamífero/ultraestructura , Uniones Intercelulares/ultraestructura , Lytechinus/embriología , Permeabilidad , Receptores Mitogénicos , Agua de Mar , Strongylocentrotus purpuratus/embriologíaRESUMEN
BACKGROUND: 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-/-). METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.