Ouabain Regulates Caveolin-1 Vesicle Trafficking by a Src-Dependent Mechanism

The Na/K-ATPase interacts with Src to form a functional receptor for endogenous cardiotonic steroids such as ouabain. The binding of ouabain to this receptor stimulates Src. We have previously shown that knockdown of Na/K-ATPase activates Src and increases scission of Caveolin-1(Cav-1) vesicle trafficking. We hypothesized that ouabain may have a hormone effect and regulate Cav-1 trafficking by Src activation. Using live cell time-lapse microscopy we observed 100nM ouabain increased scission of Cav-1 vesicles from the plasma membrane in LLC-PK1 cells in a dose-dependent manner indicating that ouabain stimulates the endocytosis of caveolae. For confirmation, we incubated cells with cholera toxin B, a protein complex that enters the cell via caveolae endocytosis. We observed cholera toxin B internalization. Next, we treated cells with Src inhibitor, PP2 and a specific antagonist of ouabain, pNaKtide and both inhibitors blocked scission of Cav-1 vesicles. In addition, we mutated Y14 to Y14F of Cav-1. We saw a decrease in scission of Cav-1 vesicles trafficking in response to ouabain stimulation. Our lab showed that by inhibiting microtubules formation reduced endocytosis in knockdown cells. Since dynamin is involved in the scission of caveolae, we tested the role of dynamin 2 (Dyn2). These studies revealed that ouabain activated Dyn2 in LLC-PK1 cells. Moreover, inhibition of Dyn2 completely obliterated ouabain-induced scission of Cav-1 vesicles. These findings provide evidence that ouabain can induce scission of Cav-1 vesicle trafficking through Na/K-ATPase by a Src dependent mechanism.