Developmental Role of Anoctamin-1/TMEM16A in Ca(2+)-Dependent Volume Change in Supporting Cells of the Mouse Cochlea

Abstract

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 Ca(2+), and cell volume change. This Ca(2+)-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; Ca2+-activated chloride channel; cell volume change; cochlea; greater epithelial ridge; supporting cell.

Fig. 1

Spontaneous activity in GER supporting cells is inhibited by anion channel blocker, NPPB. (A) Spontaneous volume change was observed in cochlea. Areas exhibiting cell volume change were indicated with white arrows in the images from before and during volume change. (B) Plot of quantification of spontaneous volume change in greater epithelial ridge (GER). During NPPB application for 20 min the change was inhibited and recovered after washout of the drug. (C) Summary bar graph shows inhibitory effect of NPPB (100 µM) on the spontaneous volume change in cochlea (n=4 each).

Fig. 2

Ano1 is expressed in the GER of the developing mouse cochlea. (A~C) Ano1 (Green) and Bestrophin-1 double immunofluorescence labeling of P10 cochlear whole-mount preparation. (D~F) Ano1 (red) and NKCC1 (green) double immunofluorescence labeling of P9 cochlear whole-mount preparation. Ano1 immunoreactivity was found in greater epithelial ridge (GER) whereas Bestrophin-1 and NKCC1 immunoreativities were limited to the interdental cell area (IDC). (G~I) Ano1 (red) and calretinin (Green) double immunofluorescence labeling of P9 cochlear whole-mount preparation. One row of inner hair cells, three rows of outer hair cells, and spiral ganglion neurons (SG) and their peripheral processes are visualized by calretinin-immunoreactivity. Ano1 Immunoreactivity is found only near inner hair cells but not near the outer hair cells. A, B, D, E, G and H are maximal projection renderings of confocal z stack slices. C, F and I are merged views of A and B, D and E, and G and H, respectively.

Fig. 3

Ano1 is expressed in GER supporting cells. (A~D) High magnification confocal images of Ano1 immunofluorescence labeling acquired from P9 cochlear whole-mount preparation. (A) Maximal projection rendering of confocal Z stack slices. (B) Single confocal slice from middle of confocal z stack in A. (C, D) The XZ (C) and YZ (D) projection images at the location marked in B with yellow vertically-crossing lines. The stereociliary bundles of the hair cells are not consistently labeled thus, stereociliary stainings shown in A and B are thought to be non-specific reaction. (E, F) Single confocal slice images of Ano1 and calretinin double immunolabeling from another P10 cochlear whole-mount preparation. (G) Merged view of E and F. There was no overlap between Ano1 and calretinin labelings.

Fig. 4

Developmental changes in Ano1 expression. Ano1 immunoreactivity in P2, P6, P10, and P16 cochlear whole-mount preparations was compared. Ano1 expression was observed in P2, P6, and P10 cochlear preparations but mostly disappeared in P16 cochlea. All images are maximal projection renderings of confocal z stack slices.

Fig. 5

Schematic diagram of spontaneous volume regulation in developmental supporting cell.