Mammalian cochlea undergoes morphological and functional changes during the postnatal period, around the hearing onset. Major changes during the initial 2 postnatal weeks of mouse include maturation of sensory hair cells and supporting cells, and acquisition of afferent and efferent innervations. During this period, supporting cells in the greater epithelial ridge (GER) of the cochlea exhibit spontaneous and periodic activities which involves ATP, increase in intracellular Ca²⁺, and cell volume change. This Ca²⁺-dependent volume change has been proposed to involve chloride channels or transporters. We found that the spontaneous volume changes were eliminated by anion channel blocker, 100 µM NPPB. Among candidates, expression of Anoctamin-1 (Ano1 or TMEM16A), bestriphin-1 and NKCC1 were investigated in whole-mount cochlea of P9-10 mice. Immunolabeling indicated high level of Ano1 expression in the GER, but not of betrophin-1 or NKCC1. Double-labeling with calretinin and confocal image analysis further elucidated the cellular localization of Ano1 immunoreactivity in supporting cells. It was tested if the Ano1 expression exhibits similar time course to the spontaneous activities in postnatal cochlear supporting cells. Cochlear preparations from P2-3, P5-6, P9-10, P15-16 mice were subjected to immunolabeling. High level of Ano1 immunoreactivity was observed in the GER of P2-3, P5-6, P9-10 cochleae, but not of P15-17 cochleae. Taken together, the localization and time course in Ano1 expression pattern correlates with the spontaneous, periodic volume changes recorded in postnatal cochlear supporting cells. From these results we propose that Ano1 is the pacemaker of spontaneous activities in postnatal cochlea.

Keywords: Anoctamin-1 (Ano1)/TMEM16A, cochlea, greater epithelial ridge, cell volume change, Ca2+-activated chloride channel, supporting cell