Ammonium ion substitutes for K+ in ATP-dependent Na+ transport by basolateral membrane vesicles

Ion-transporting cells from posterior gills of blue crabs (Callinectes sapidus) acclimated to low salinity were used as starting material for the preparation of microsomal membrane vesicles by density gradient centrifugation. The Na+-K+-adenosinetriphosphatase (ATPase)-enriched basolateral vesicles were loaded with KCl- or NH4+-containing medium by dilution and centrifugation, and initial rates of 22Na+ uptake into the vesicles were measured by a rapid filtration procedure. Varying the extravesicular sucrose concentration altered equilibrium uptake of 22Na+, indicating the existence of osmotically sensitive vesicles. Monensin, a sodium-specific ionophore, enhanced passive uptake of 22Na+ across the vesicle membrane in the absence of ATP. With 100 mM KCl in the intravesicular medium, addition of ATP to the extravesicular medium increased initial rates of 22Na+ uptake 10- to 20-fold over levels measured without ATP. A nonhydrolyzable ATP analog failed to stimulate 22Na+ uptake. Intravesicular K+ could be replaced by NH4+ but not by choline. With NH4+ as counterion, Na+ transport was inhibited by digitoxin, but valinomycin had no effect. A study of the kinetic effects of intravesicular K+ and NH4+ on initial rates of 22Na+ uptake indicated the existence of two classes of binding sites, one responding to counterion concentrations in the millimolar range and a second class responding to counterion concentrations over 50 mM. The results indicate that ATP-dependent 22Na+ uptake by membrane vesicles from Callinectes sapidus gill, mediated by Na+-K+-ATPase, can utilize either K+ or NH4+ as counterion