Receptor-mediated inhibition of renal Na⁺-K⁺-ATPase is associated with endocytosis of its α- and β-subunits

The mechanisms involved in receptor-mediated inhibition of Na⁺ -K⁺ - ATPase remain poorly understood. In this study, we evaluate whether inhibition of proximal tubule Na⁺ -K⁺ -ATPase activity by dopamine is linked to its removal from the plasma membrane and internalization into defined intracellular compartments. Clathrin-coated vesicles were isolated by sucrose gradient centrifugation and negative lectin selection, and early and late endosomes were separated on a flotation gradient. Inhibition of Na⁺- K⁺-ATPase activity by dopamine, in contrast to its inhibition by ouabain, was accompanied by a sequential increase in the abundance of the α-subunit in clathrin-coated vesicles (1 min), early endosomes (2.5 min), and late endosomes (5 min), suggesting its stepwise translocation between these organelles. A similar pattern was found for the β-subunit. The increased incorporation of both subunits in all compartments was blocked by calphostin C. The results demonstrate that the dopamine-induced decrease in Na⁺- K⁺ATPase activity in proximal tubules is associated with internalization of its α- and β-subunits into early and late endosomes via a clathrin- dependent pathway and that this process is protein kinase C dependent. The presence of Na⁺-K⁺ATPase subunits in endosomes suggests that these compartments may constitute normal traffic reservoirs during pump degradation and/or synthesis.