Calcium distribution and mobilization during depolarization in single cochlear hair cells. Imaging microscopy and fura-2.

Intracellular distribution of cytoplasm-free Ca2+ concentrations ((Ca2+)i) and dynamic changes during stimulation of viable hair cells were studied using digital imaging microscopy and the Ca2(+)-sensitive dye fura-2. (Ca2+)i was visualized on pseudo-colour images and three-dimensional computer graphics. In the resting state, the intra-cellular distribution of (Ca2+)i in both the outer and inner hair cells was heterogeneous, and the amount of (Ca2+)i in most of the peripheral cytoplasm just beneath the plasma membrane was greater than that throughout the entire cytoplasm. Cell depolarization, induced by elevated K+, led to an increase in (Ca2+)i in the outer hair cells. The increase in (Ca2+)i was not observed under conditions of depolarization in Ca2(+)-free medium. These observations are interpreted to mean that the increase in (Ca2+)i is induced by depolarization with the result that there is an influx of extracellular Ca2+ into the cytoplasm. When Mn2+ was applied during depolarization, a fluorescence quenching occurred. By such means the site of Ca2+ channels was elucidated.