Quantitative localization of Na-K pump site in frog inner ear dark cells

The Na-K pump site distribution within the dark cells of the frog inner ear was examined with quantitative freeze-dry [3H]ouabain autoradiography. Control experiments revealed that ouabain binding was specific. Na-K pumps were located in the basolateral dark cell plasma membrane and were distributed nonuniformly across the epithelial apical-basal axis. The highest concentration (sites per volume) was found over the basal region and the lowest over the apical region. The average pump site concentration for dark cells from four animals was 25 X 10(3) sites/micron3. Stereological analysis of conventionally fixed tissue revealed that the plasma membrane area per volume (Sv) was also nonuniform across the apical-basal axis with the highest surface to volume ratio in the basal region. The average Sv for two dark cell regions was 5.5 micron2/micron3. Combination of autoradiography and stereology revealed that the pump site density (sites per membrane area) was independent of position along the apical-basal axis and was equal to 4500 +/- 25%, a value close to the upper limit as determined by the diameter of the isolated Na-K-ATPase.