Ca2+-dependent, temperature-sensitive regulation of Na+ channels in tight epithelia. A study using membrane vesicles

Na+ fluxes were measured in toad bladder microsomes. Under favorable conditions, 60-90% of the tracer uptake was blocked by amiloride (Ki = 2.3 X 10(-8) M), i.e. mediated by the apical Na+-specific channels. Vesicles derived from cells maintained at 0 degrees C exhibited relatively small amiloride-sensitive fluxes. However, incubating the scraped cells at 25 degrees C prior to homogenization induced a nearly 5-fold increase of the amiloride-blockable flux in vesicles. This activation was fairly slow (t 1/2 = 5-10 min), irreversible, and strongly dependent on the incubation temperature. On the other hand, the Na+-specific apical conductance measured in mounted bladders was only slightly affected by the incubation temperature. The above activation process could be observed only in Ca2+-free EGTA-containing solutions. Adding Ca2+ (1 mM) to the cell suspension and subsequently removing it before homogenization blocked almost completely the amiloride-sensitive tracer uptake in the vesicles. The data are compatible with the model that the epithelial Na+ channels are down-regulated by a Ca2+-dependent reaction. The incubation of scraped, somewhat permeabilized cells in a Ca2+-free solution releases channels from this down-regulation and increases the Na+ conductance in a temperature-dependent process. The regulation of channels appears to involve a cytoplasmic factor which induces a stable modification of the apical membrane, preserved by the isolated vesicle.