Binding of [3H]Ouabain to Split Frog Skin

A s s T R A C X The binding of [SH]ouabain to the serosal side was studied in a chambered preparat ion of frog skin, free of connective tissue, while the short circuit (I~) was concurrent ly monitored. Both ouabain binding and 1~ inhibition proceeded as hyperbolic functions of time. A plot of the number of ouabain molecules bound vs. the corresponding values of Iae inhibition (percent) yielded a straight line, yet showed that one-third of the binding occurred before any inhibition of Ise. Upon separat ion of the skins into two groups based upon initial I se( l~l ) -h igh, >20 /~A/cm 2 and low, <10 /~A/cm 2, we observed two distinct populations. The high I~t skins bound very little ouabain before inhibition of lsc whereas low Isel skins bound one-half of the total number o f sites before exhibit ing any inhibition o f l ~ . These observations strongly suggest that (a) the Na,K-ATPase is directly involved in the generation of Ise, and (b) at low Ise, inhibition of some pumps by ouabain causes a "recruitment" of other pumps to increase their turnover rate and maintain Ise relatively unaffected. In addit ion, the binding of ouabain also displayed various characteristics that were consistent with known propert ies o f the Na,K-ATPase: (a) increased intracellular K/Na concentrations, whether achieved th rough the addit ion of amiloride or removal of Na from the outside medium, led to a significant decrease in ouabain binding rate relative to paired controls; and (b) ouabain binding, ei ther with normal or decreased intracellular Na, was significantly reduced in the presence of elevated K in the serosal bathing medium. Finally, the number o f ouabain molecules bound to the frog skins was not correlated with their initial Ise values, indicating that the spontaneous skin-to-skin variation in lae was not related to the number of functional pump sites but, ra ther , to their turnover rate.