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Despite the crucial role of endolymph as an electrochemical motor for mechanotransduction, little is known about the processes that govern endolymph formation. To address these, we took advantage of an organotypic rodent model, which regenerates a genuine neonatal vestibular endolymphatic compartment, facilitating the determination of endolymphatic [K<jats:sup>+<\/jats:sup>] and transepithelial potential (Vt) during endolymph formation. While mature Vt levels are almost immediately achieved, K<jats:sup>+<\/jats:sup>accumulates to reach a steady [K<jats:sup>+<\/jats:sup>] by day 5 in culture. Inhibition of sensory cell K<jats:sup>+<\/jats:sup>efflux enhances [K<jats:sup>+<\/jats:sup>] regardless of the blocker used (FM1.43, amikacin, gentamicin, or gadolinium). Targeting K<jats:sup>+<\/jats:sup>secretion with bumetanide partially and transiently reduces [K<jats:sup>+<\/jats:sup>], while ouabain application and<jats:italic>Kcne1<\/jats:italic>deletion almost abolishes it. Immunofluorescence studies demonstrate that dark cells do not express Na-K-2Cl cotransporter 1 (the target of bumetanide) in cultured and young mouse utricles, while Na\/K-ATPase (the target of ouabain) is found in dark cells and transitional cells. This global analysis of the involvement of endolymphatic homeostasis actors in the immature organ (1) confirms that KCNE1 channels are necessary for K<jats:sup>+<\/jats:sup>secretion, (2) highlights Na\/K-ATPase as the key endolymphatic K<jats:sup>+<\/jats:sup>provider and shows that Na-K-2Cl cotransporter 1 has a limited impact on K<jats:sup>+<\/jats:sup>influx, and (3) demonstrates that transitional cells are involved in K<jats:sup>+<\/jats:sup>secretion in the early endolymphatic compartment.<\/jats:p>","DOI":"10.1523\/jneurosci.2430-11.2011","type":"journal-article","created":{"date-parts":[[2011,11,17]],"date-time":"2011-11-17T09:16:37Z","timestamp":1321521397000},"page":"16541-16549","source":"Crossref","is-referenced-by-count":35,"title":["Critical Roles of Transitional Cells and Na\/K-ATPase in the Formation of Vestibular 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