Regulation of sodium pump endocytosis by cardiotonic steroids: Molecular mechanisms and physiological implications

Abstract

We have previously shown that ouabain and other cardiotonic steroids interact with the plasmalemmal Na/K-ATPase and cause a time and dose dependent endocytosis of the Na/K-ATPase. This endocytosis is demonstrable using fluorescence imaging as well as conventional biochemical and biophysical cell separation methods. In proximal tubule cells, this process appears to regulate the density of basolateral Na/K-ATPase expression directly as well as indirectly modulate transepithelial sodium transport. Work with genetic manipulations, as well as pharmacological agents with cell culture models, have demonstrated that the cardiotonic steroid stimulated endocytosis of the plasmalemmal Na/K-ATPase requires caveolin and clathrin as well as the activation of c-Src, transactivation of the EGFR and activation of PI3K. Interestingly c-Src, EGFR and ERK1/2 all appear to be endocytosed along with the plasmalemmal Na/K-ATPase. These observations suggest a close analogy between a subset of plasmalemmal Na/K-ATPase and signaling companions with conventional receptor tyrosine kinases. While further studies are necessary to delineate the role of this endocytosis in the generation as well as the limit of signal transduction through the Na/K-ATPase signal cascade, we propose that it has an important role in the regulation of renal sodium handling as well as other important processes.

Figure 1

Schematic illustration of Na/K-ATPase α1 subunit, caveolin-1 and c-Src, A: In the Na/K-ATPase α1 subunit, caveolin-binding motif (CBM) is located in the cytosolic side of N-terminus, proximally to TM1 (transmembrane domain 1). CD2 is the first cytosolic loop between TM2 and TM3, bearing activation domain (AD). CD3 is the second cytosolic loop between TM4 and TM5, bearing nucleotide-binding domain (ND) and phosphorylation domain (PD) B; In caveolin, C-terminal and N-terminal membrane attachment domains (C-MAD and N-MAD) are important for membrane attachment. The scaffolding domain (SD) is overlapped with N-MAD and oligomerization domain (OD). The transmembrane domain (TMD) is for membrane insertion. C: c-Src is a cytosolic kinase, containing SH3, SH2, and kinase (N-lobe and C-lobe) domains. C-terminal tail Y527 is the negative regulatory phosphorylation site, and Y416 (bearing in the A-loop helix between kinase domain N-lobe and C-lobe) is the major autophosphorylation site.

Figure 2

Schematic illustration ouabain-induced endocytosis of the Na/K-ATPase and NHE3. Binding of ouabain to the Na/K-ATPase α1 activates c-Src and consequent signal cascades. This process also recruits PI3K P85α subunit, AP-2, and clathrin to the α1 subunit as well as promotes the formation of clathrin-coated pits. In response to ouabain, the Na/K-ATPase is internalized into clathrin-coated vesicles (CCV), early endosomes (EE), and late endosomes (LE). Some important signaling molecules (EGFR, c-Src, and ERK1/2) are also accumulated in CCV, EE, and LE. Interestingly, ouabain-activated signaling also induces endocytosis or downregulation of NHE3. The ultimately effect is reduced transepithelial sodium reabsorption.