Localization of intracellular compartments that exchange Na,K-ATPase molecules with the plasma membrane in a hormone-dependent manner.

BACKGROUND AND PURPOSE: Dopamine is a major regulator of sodium reabsorption in proximal tubule epithelia. By binding to D1-receptors, dopamine induces endocytosis of plasma membrane Na,K-ATPase, resulting in a reduced capacity of the cells to transport sodium, thus contributing to natriuresis. We have previously demonstrated several aspects of the molecular mechanism by which dopamine induces Na,K-ATPase endocytosis; however, the location of intracellular compartments containing Na,K-ATPase molecules has not been identified. EXPERIMENTAL APPROACH: In this study, we used different approaches to determine the localization of Na,K-ATPase-containing intracellular compartments. By expression of fluorescent-tagged Na,K-ATPase molecules in opossum kidney cells, a cell culture model of proximal tubule epithelia, we used fluorescence microscopy to determine cellular distribution of the fluorescent molecules and the effects of dopamine on this distribution. By labelling cell surface Na,K-ATPase molecules from the cell exterior with either biotin or an epitope-tagged antibody, we determined the localization of the tagged Na,K-ATPase molecules after endocytosis induced by dopamine. KEY RESULTS: In cells expressing fluorescent-tagged Na,K-ATPase molecules, there were intracellular compartments containing Na,K-ATPase molecules. These compartments were in very close proximity to the plasma membrane. Upon treatment of the cells with dopamine, the fluorescence labelling of these compartments was increased. The labelling of these compartments was also observed when the endocytosis of biotin- or antibody-tagged plasma membrane Na,K-ATPase molecules was induced by dopamine. CONCLUSIONS AND IMPLICATIONS: The intracellular compartments containing Na,K-ATPase molecules are located just underneath the plasma membrane.

Cytoplasmic segment interactions in the alpha1-subunit of the rat Na+, K+-atpase.

The currently accepted topographical model for the organization of the alpha-subunit of the Na+, K+-ATPase in the membrane considers that the protein has ten transmembrane segments and six cytoplasmic loops. Evidence of interaction between the cytoplasmic regions may contribute to a better understanding of the structure/function relationship of this protein. In this study, the first four cytoplasmic segments (C1, C2, C3 and C4) of the rat alpha1 subunit were expressed in Escherichia Coli. The large cytoplasmic loop between transmembrane segments four and five (C3) retained its native structure as demonstrated by the ability of ATP to protect against chemical modification by Fluorescein 5-isothiocyanate (FITC). Interaction studies were conducted by an overlay assay (Far Western blots) and surface plasmon resonance technology. We observed that C3 interacts with the N-terminal segment of the Na+, K+-ATPase, C1; and that both C1 and C3 interact with the cytoplasmic segments C2 and C4.

Simultaneous phosphorylation of Ser11 and Ser18 in the alpha-subunit promotes the recruitment of Na(+),K(+)-ATPase molecules to the plasma membrane.

Renal sodium homeostasis is a major determinant of blood pressure and is regulated by several natriuretic and antinatriuretic hormones. These hormones, acting through intracellular second messengers, either activate or inhibit proximal tubule Na(+),K(+)-ATPase. We have shown previously that phorbol ester (PMA) stimulation of endogenous PKC leads to activation of Na(+),K(+)-ATPase in cultured proximal tubule cells (OK cells) expressing the rodent Na(+), K(+)-ATPase alpha-subunit. We have now demonstrated that the treatment with PMA leads to an increased amount of Na(+),K(+)-ATPase molecules in the plasmalemma, which is proportional to the increased enzyme activity. Colchicine, dinitrophenol, and potassium cyanide prevented the PMA-dependent stimulation of activity without affecting the increased level of phosphorylation of the Na(+), K(+)-ATPase alpha-subunit. This suggests that phosphorylation does not directly stimulate Na(+),K(+)-ATPase activity; instead, phosphorylation may be the triggering mechanism for recruitment of Na(+),K(+)-ATPase molecules to the plasma membrane. Transfected cells expressing either an S11A or S18A mutant had the same basal Na(+),K(+)-ATPase activity as cells expressing the wild-type rodent alpha-subunit, but PMA stimulation of Na(+),K(+)-ATPase activity was completely abolished in either mutant. PMA treatment led to phosphorylation of the alpha-subunit by stimulation of PKC-beta, and the extent of this phosphorylation was greatly reduced in the S11A and S18A mutants. These results indicate that both Ser11 and Ser18 of the alpha-subunit are essential for PMA stimulation of Na(+), K(+)-ATPase activity, and that these amino acids are phosphorylated during this process. The results presented here support the hypothesis that PMA regulation of Na(+),K(+)-ATPase is the result of an increased number of Na(+),K(+)-ATPase molecules in the plasma membrane.

Phosphoinositide-3 kinase binds to a proline-rich motif in the Na+, K+-ATPase alpha subunit and regulates its trafficking.

Endocytosis of Na(+),K(+)-ATPase molecules in response to G protein-coupled receptor stimulation requires activation of class I(A) phosphoinositide-3 kinase (PI3K-I(A)) in a protein kinase C-dependent manner. In this paper, we report that PI3K-I(A), through its p85alpha subunit-SH3 domain, binds to a proline-rich region in the Na(+),K(+)-ATPase catalytic alpha subunit. This interaction is enhanced by protein kinase C-dependent phosphorylation of a serine residue that flanks the proline-rich motif in the Na(+),K(+)-ATPase alpha subunit and results in increased PI3K-I(A) activity, an effect necessary for adaptor protein 2 binding and clathrin recruitment. Thus, Ser-phosphorylation of the Na(+),K(+)-ATPase catalytic subunit serves as an anchor signal for regulating the location of PI3K-I(A) and its activation during Na(+),K(+)-ATPase endocytosis in response to G protein-coupled receptor signals.

G protein-coupled receptors regulate Na+,K+-ATPase activity and endocytosis by modulating the recruitment of adaptor protein 2 and clathrin.

Inhibition of Na(+),K(+)-ATPase (NKA) activity in renal epithelial cells by activation of G protein-coupled receptors is mediated by phosphorylation of the catalytic alpha-subunit followed by endocytosis of active molecules. We examined whether agonists that counteract this effect do so by dephosphorylation of the alpha-subunit or by preventing its internalization through a direct interaction with the endocytic network. Oxymetazoline counteracted the action of dopamine on NKA activity, and this effect was achieved not by preventing alpha-subunit phosphorylation, but by impaired endocytosis of alpha-subunits into clathrin vesicles and early and late endosomes. Dopamine-induced inhibition of NKA activity and alpha-subunit endocytosis required the interaction of adaptor protein 2 (AP-2) with the catalytic alpha-subunit. Phosphorylation of the alpha-subunit is essential because dopamine failed to promote such interaction in cells lacking the protein kinase C phosphorylation residue (S18A). Confocal microscopy confirmed that oxymetazoline prevents incorporation of NKA molecules into clathrin vesicles by inhibiting the ability of dopamine to recruit clathrin to the plasma membrane. Dopamine decreased the basal levels of inositol hexakisphosphate (InsP(6)), whereas oxymetazoline prevented this effect. Similar increments (above basal) in the concentration of InsP(6) induced by oxymetazoline prevented AP-2 binding to the NKA alpha-subunit in response to dopamine. In conclusion, inhibition of NKA activity can be reversed by preventing its endocytosis without altering the state of alpha-subunit phosphorylation; increased InsP(6) in response to G protein-coupled receptor signals blocks the recruitment of AP-2 and thereby clathrin-dependent endocytosis of NKA.

PKC-beta and PKC-zeta mediate opposing effects on proximal tubule Na+,K+-ATPase activity.

Dopamine (DA) inhibits rodent proximal tubule Na+,K+-ATPase via stimulation of protein kinase C (PKC). However, direct stimulation of PKC by phorbol 12-myristate 13-acetate (PMA) results in increased Na+,K+-ATPase. LY333531, a specific inhibitor of the PKC-beta isoform, prevents PMA-dependent activation of Na+,K+-ATPase, but has no effect on DA inhibition of this activity. A similar result was obtained with a PKC-beta inhibitor peptide. Concentrations of staurosporine, that inhibits PKC-zeta, prevent DA-dependent inhibition of Na+,K+-ATPase and a similar effect was obtained with a PKC-zeta inhibitor peptide. Thus, PMA-dependent stimulation of Na+,K+-ATPase is mediated by activation of PKC-beta, whereas inhibition by DA requires activation of PKC-zeta.

Dopamine-induced endocytosis of Na+,K+-ATPase is initiated by phosphorylation of Ser-18 in the rat alpha subunit and Is responsible for the decreased activity in epithelial cells.

Dopamine inhibits Na+,K+-ATPase activity in renal tubule cells. This inhibition is associated with phosphorylation and internalization of the alpha subunit, both events being protein kinase C-dependent. Studies of purified preparations, fusion proteins with site-directed mutagenesis, and heterologous expression systems have identified two major protein kinase C phosphorylation residues (Ser-11 and Ser-18) in the rat alpha1 subunit isoform. To identify the phosphorylation site(s) that mediates endocytosis of the subunit in response to dopamine, we have performed site-directed mutagenesis of these residues in the rat alpha1 subunit and expressed the mutated forms in a renal epithelial cell line. Dopamine inhibited Na+,K+-ATPase activity and increased alpha subunit phosphorylation and clathrin-dependent endocytosis into endosomes in cells expressing the wild type alpha1 subunit or the S11A alpha1 mutant, and both effects were blocked by protein kinase C inhibition. In contrast, dopamine did not elicit any of these effects in cells expressing the S18A alpha1 mutant. While Ser-18 phosphorylation is necessary for endocytosis, it does not affect per se the enzymatic activity: preventing endocytosis with wortmannin or LY294009 blocked the inhibitory effect of dopamine on Na+,K+-ATPase activity, although it did not alter the increased alpha subunit phosphorylation induced by this agonist. We conclude that dopamine-induced inhibition of Na+, K+-ATPase activity in rat renal tubule cells requires endocytosis of the alpha subunit into defined intracellular compartments and that phosphorylation of Ser-18 is essential for this process.

Phosphorylation of the catalyic alpha-subunit constitutes a triggering signal for Na+,K+-ATPase endocytosis.

Inhibition of Na+,K+-ATPase activity by dopamine is an important mechanism by which renal tubules modulate urine sodium excretion during a high salt diet. However, the molecular mechanisms of this regulation are not clearly understood. Inhibition of Na+,K+-ATPase activity in response to dopamine is associated with endocytosis of its alpha- and beta-subunits, an effect that is protein kinase C-dependent. In this study we used isolated proximal tubule cells and a cell line derived from opossum kidney and demonstrate that dopamine-induced endocytosis of Na+,K+-ATPase and inhibition of its activity were accompanied by phosphorylation of the alpha-subunit. Inhibition of both the enzyme activity and its phosphorylation were blocked by the protein kinase C inhibitor bisindolylmaleimide. The early time dependence of these processes suggests a causal link between phosphorylation and inhibition of enzyme activity. However, after 10 min of dopamine incubation, the alpha-subunit was no longer phosphorylated, whereas enzyme activity remained inhibited due to its removal from the plasma membrane. Dephosphorylation occurred in the late endosomal compartment. To further examine whether phosphorylation was a prerequisite for subunit endocytosis, we used the opossum kidney cell line transfected with the rodent alpha-subunit cDNA. Treatment of this cell line with dopamine resulted in phosphorylation and endocytosis of the alpha-subunit with a concomitant decrease in Na+,K+-ATPase activity. In contrast, none of these effects were observed in cells transfected with the rodent alpha-subunit that lacks the putative protein kinase C-phosphorylation sites (Ser11 and Ser18). Our results support the hypothesis that protein kinase C-dependent phosphorylation of the alpha-subunit is essential for Na+,K+-ATPase endocytosis and that both events are responsible for the decreased enzyme activity in response to dopamine.

Stimulation of protein kinase C rapidly reduces intracellular Na+ concentration via activation of the Na+ pump in OK cells.

Na+ reabsorption is regulated in proximal tubules by hormones that stimulate protein kinase C (PKC). To determine whether stimulation of PKC causes a reduction in intracellular Na+ concentration ([Na+]i) that might link Na+ pump activation to increased Na+ reabsorption, [Na+]i was measured in kidney cells loaded with the Na+-sensitive fluorescent indicator SBFI. Rapid digital imaging fluorescence microscopy determinations were performed in epithelial kidney cells transfected with the rodent Na+ pump alpha1 cDNA. In 42 determinations, the basal [Na+]i was 19.7 +/- 2.4 mM. Stimulation of PKC reduced the [Na+]i to 5.6 +/- 0.6 mM in approximately 10 sec. This drastic change in [Na+]i requires a transient 74-120-fold increase in Na+ pump activity. After the new steady state [Na+]i is reached, the Na+ pump is 58% activated. The entry of Na+ into the cells is not affected by stimulation of PKC; therefore, the reduction in [Na+]i is exclusively dependent on activation of the Na+ pump. Accordingly, PKC stimulation does not affect the [Na+]i of cells expressing a mutant Na+ pump that is not stimulated by PKC. The decrease in [Na+]i observed in cells transfected with the rodent Na+ pump alpha1 cDNA is large and sufficiently fast that it is expected to stimulate rapidly passive Na+-influx into the cells, thereby accounting for the observed PKC-induced stimulation of Na+ reabsorption.

Regulation of Na,K-ATPase transport activity by protein kinase C.

Considerable evidence indicates that the renal Na+,K+-ATPase is regulated through phosphorylation/dephosphorylation reactions by kinases and phosphatases stimulated by hormones and second messengers. Recently, it has been reported that amino acids close to the NH2-terminal end of the Na+,K+-ATPase alpha-subunit are phosphorylated by protein kinase C (PKC) without apparent effect of this phosphorylation on Na+,K+-ATPase activity. To determine whether the alpha-subunit NH2-terminus is involved in the regulation of Na+, K+-ATPase activity by PKC, we have expressed the wild-type rodent Na+,K+-ATPase alpha-subunit and a mutant of this protein that lacks the first thirty-one amino acids at the NH2-terminal end in opossum kidney (OK) cells. Transfected cells expressed the ouabain-resistant phenotype characteristic of rodent kidney cells. The presence of the alpha-subunit NH2-terminal segment was not necessary to express the maximal Na+,K+-ATPase activity in cell membranes, and the sensitivity to ouabain and level of ouabain-sensitive Rb+-transport in intact cells were the same in cells transfected with the wild-type rodent alpha1 and the NH2-deletion mutant cDNAs. Activation of PKC by phorbol 12-myristate 13-acetate increased the Na+,K+-ATPase mediated Rb+-uptake and reduced the intracellular Na+ concentration of cells transfected with wild-type alpha1 cDNA. In contrast, these effects were not observed in cells expressing the NH2-deletion mutant of the alpha-subunit. Treatment with phorbol ester appears to affect specifically the Na+,K+-ATPase activity and no evidence was observed that other proteins involved in Na+-transport were affected. These results indicate that amino acid(s) located at the alpha-subunit NH2-terminus participate in the regulation of the Na+,K+-ATPase activity by PKC.