Subcellular distribution of protein kinase C in the living outer hair cell of the guinea pig cochlea

Abstract

Immunohistochemical staining using isoform-specific antibodies and intracellular localization using fluorescent probes for protein kinase C (PKC) were evaluated in the cochlear outer hair cell (OHC). Among three isoforms of classic PKC, PKC α was selectively stained in the fixed OHC as well as inner hair cells under a surface preparation method. Two types of fluorescent probes to detect subcellular localization of PKC were observed with a confocal laser scanning microscopy in the present study, fim-1 diacetate which binds to the ATP-competitive catalytic domain of PKC and Bodipy FL C 12-phorbol acetate which binds to specific site localized to the first cysteine-rich loop of the C1 region in the regulatory domain. High fluorescence intensity of both dyes was observed in subcuticular and subsynaptic regions, infracuticular network, and along the lateral wall. The displacement experiments to evaluate binding specificity were performed by incubating Bodipy FL C 12-phorbol acetate in the presence of 10 μM phorbol 12-myritate 13-acetate (PMA) and the fluorescence was totally disappeared. For the acute treatment of phorbol ester, cells were preincubated with 1 μM PMA 30 min before loading with fim-1 diacetate. The brightest area in the plasma membrane became much larger as compared with untreated cells, which suggests a dramatic translocation of PKC to the plasma membrane. The biological functions involving PKC in the OHC are discussed.