Elsevier

Neuroscience

Volume 130, Issue 2, 2005, Pages 475-484
Neuroscience

Localization and developmental expression of BK channels in mammalian cochlear hair cells

https://doi.org/10.1016/j.neuroscience.2004.09.038Get rights and content

Abstract

The expression of Slo channels (α subunits of BK channels) was investigated in the developing mouse cochlea using a polyclonal antibody against the C-terminal part of the protein (residues 1098–1196). The first BK channel immunoreactivity was observed in the cochlea at E18, where it was localized within the cytoplasm of cells lining the area of the organ of Corti and the spiral ganglion. There was an increase of immunoreactivity in all cells bordering the scala media (supporting and hair cells of the organ of Corti, the stria vascularis and the Reissner's membrane) in the following stages (postnatal day [P] 0 and P6). From P12 to adult, a strong membranous labeling, increasing with age, appeared in inner hair cells. The distribution of BK channels was mainly observed as dense elongated plaques localized in the lateral membrane below the cuticular plate. In addition, a more discrete immunolabeling for BK channels, as punctuated dots, was observed in the synaptic area of inner hair cells. This dual localization of BK channels within inner hair cells was confirmed by a different technique using a fluorescently labeled high-affinity ligand of these channels: IbTX-D19C-Alexa488. We demonstrated under patch clamp experiments that this fluorescent toxin conserved its native property, i.e. to reversibly inhibit BK currents in isolated inner hair cells. The fluorescent toxin, both in living or fixed tissues, also showed a preferential binding to mature inner hair cells with a similar subcellular distribution described above using immunocytochemical technique. Overall, our present results confirm the appearance of membranous BK channels around P12 in mouse inner hair cells, an age at which the auditory system becomes functional. The expression of BK channels in mature inner hair cells, near the site of mechanical-transduction, might serve to limit receptor potential attenuation due to the space constant, and thus permitting these sensory cells to function as fast and sensitive transducers.

Section snippets

Immunohistochemistry

C57BL/6J mice at embryonic stages (E) 15–16 and 18, P0, P6, P12, P15 and adult were used for the present study. Minimums of five animals were used per stage. Mice were killed by cervical dislocation (adult) and/or were then decapitated. Animals were deeply anaesthetized before being killed and their handling throughout these experiments was performed with authorisation of the French Ministry of Agriculture and in accordance with EEC regulations. All efforts were made to minimize the number of

Distribution of BK channels in the adult mouse cochlea

Immunoreactivity of Slo channels was observed in various structures of the mature cochlea. Mid to strong expression was noted in the spiral ligament and in the surface cells of the spiral prominence (Fig. 1a). A strong immunoreactivity was also observed in Böttcher cells located on the pars pectinata of the basilar membrane and in Claudius cells (Fig. 1a–c). In the organ of Corti, a strong expression was observed in IHCs (Fig. 1a, b). We could identify no evidence for a gradient of differential

Discussion

Our study clearly shows that mature IHCs of the mouse cochlea strongly express the Slo-encoded α-subunit protein, known to constitute when assembled in tetramers functional BK channels (Atkinson et al., 1991). These results are in agreement with recent studies showing that adult rat IHCs express Slo transcripts mRNA (Brandle et al., 2001; Skinner et al., 2003; Langer et al., 2003). They also fit well with recent studies showing strong immunolabeling of BK channels in mature guinea-pig or mouse

Acknowledgments

We are grateful to Professor James Saunders for his comments and English correction of the manuscript.

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