Characteristics of the entry process for sodium in transporting epithelia as revealed with amiloride

1. The permeation of sodium ions trhough the mucosal surface of frog skin epithelium at different transepithelial potentials has been investigated using the blocking drug amiloride. 2. An increase in serosal negativity in voltage-clamped skins was associated with an increase in the absolute amount of inhibition caused by a fixed concentration of amiloride. Hyperpolarizing or depolarizing skins with respect to the short-circuited condition did not affect the apparent affinity of amiloride for the entry sites. 3. When skins were voltage clamped at -50 mV (serosa negative) the specific binding of amiloride to sodium entry sites was increased by 77% compared to the short-circuited condition. Skins clamped at +50 mV had only 72% of the specific binding found in short-circuited skins. Experiments with a second blocking drug, triamterene, indicated that the extra binding sites appearing at -50mV were similar to those found under short-circuit conditions. The appearance and disappearance of binding sites may reflect changes in cell volume. 4. The findings suggest that the increased sodium current which flows when skins are clamped at -50 mV results from an increase in the number of entry sites, and perhaps also to a voltage sensitive increase in flux through each entry site.