Riad Efendiev

Hypertension-Linked Mutation in the Adducin α-Subunit Leads to Higher AP2-μ2 Phosphorylation and Impaired Na+,K+-ATPase Trafficking in Response to GPCR Signals and Intracellular Sodium

&agr;-Adducin polymorphism in humans is associated with abnormal renal sodium handling and high blood pressure. The mechanisms by which mutations in adducin affect the renal set point for sodium excretion are not known. Decreases in Na+,K+-ATPase activity attributable to endocytosis of active units in renal tubule cells by dopamine regulates sodium excretion during high-salt diet. Milan rats carrying the hypertensive adducin phenotype have a higher renal tubule Na+,K+-ATPase activity, and their Na+,K+-ATPase molecules do not undergo endocytosis in response to dopamine as do those of the normotensive strain. Dopamine fails to promote the interaction between adaptins and the Na+,K+-ATPase because of adaptin-&mgr;2 subunit hyperphosphorylation. Expression of the hypertensive rat or human variant of adducin into normal renal epithelial cells recreates the hypertensive phenotype with higher Na+,K+-ATPase activity, &mgr;2-subunit hyperphosphorylation, and impaired Na+,K+-ATPase endocytosis. Thus, increased renal Na+,K+-ATPase activity and altered sodium reabsorption in certain forms of hypertension could be attributed to a mutant form of adducin that impairs the dynamic regulation of renal Na+,K+-ATPase endocytosis in response to natriuretic signals.

PKC-β and PKC-ζ 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‐β 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‐β inhibitor peptide. Concentrations of staurosporine, that inhibits PKC‐ζ, prevent DA‐dependent inhibition of Na+,K+‐ATPase and a similar effect was obtained with a PKC‐ζ inhibitor peptide. Thus, PMA‐dependent stimulation of Na+,K+‐ATPase is mediated by activation of PKC‐β, whereas inhibition by DA requires activation of PKC‐ζ.

Tyrosine 537 within the Na+,K+-ATPase α-Subunit Is Essential for AP-2 Binding and Clathrin-dependent Endocytosis

In renal epithelial cells endocytosis of Na+,K+-ATPase molecules is initiated by phosphorylation of its α1-subunit, leading to activation of phosphoinositide 3-kinase and adaptor protein-2 (AP-2)/clathrin recruitment. The present study was performed to establish the identity of the AP-2 recognition domain(s) within the Na+,K+-ATPase α1-subunit. We identified a conserved sequence (Y537LEL) within the α1-subunit that represents an AP-2 binding site. Binding of AP-2 to the Na+,K+-ATPase α1-subunit in response to dopamine (DA) was increased in OK cells stably expressing the wild type rodent α-subunit (OK-WT), but not in cells expressing the Y537A mutant (OK-Y537A). DA treatment was associated with increased α1-subunit abundance in clathrin vesicles from OK-WT but not from OK-Y537A cells. In addition, this mutation also impaired the ability of DA to inhibit Na+,K+-ATPase activity. Because phorbol estersincrease Na+,K+-ATPase activity in OK cells, and this effect was not affected by the Y537A mutation, the present results suggest that the identified motif is specifically required for DA-induced AP-2 binding and Na+,K+-ATPase endocytosis.

Agonist-dependent Regulation of Renal Na+,K+-ATPase Activity Is Modulated by Intracellular Sodium Concentration

We tested the hypothesis that the level of intracellular sodium modulates the hormonal regulation of the Na+,K+-ATPase activity in proximal tubule cells. By using digital imaging fluorescence microscopy of a sodium-sensitive dye, we determined that the sodium ionophore monensin induced a dose-specific increase of intracellular sodium. A correspondence between the elevation of intracellular sodium and the level of dopamine-induced inhibition of Na+,K+-ATPase activity was determined. At basal intracellular sodium concentration, stimulation of cellular protein kinase C by phorbol 12-myristate 13-acetate (PMA) promoted a significant increase in Na+,K+-ATPase activity; however, this activation was gradually reduced as the concentration of intracellular sodium was increased to become a significant inhibition at concentrations of intracellular sodium higher than 16 mm. Under these conditions, PMA and dopamine share the same signaling pathway to inhibit the Na+,K+-ATPase. The effects of PMA and dopamine on the Na+,K+-ATPase activity and the modulation of these effects by different intracellular sodium concentrations were not modified when extracellular and intracellular calcium were almost eliminated. These results suggest that the level of intracellular sodium modulates whether hormones stimulate, inhibit, or have no effect on the Na+,K+-ATPase activity leading to a tight control of sodium reabsorption.

Hormonal‐dependent recruitment of Na+,K+‐ATPase to the plasmalemma is mediated by PKCβ and modulated by [Na+]i

The present study demonstrates that stimulation of hormonal receptors of proximal tubule cells with the serotonin‐agonist 8‐hydroxy‐2‐(di‐n‐propylamino) tetraline (8‐OH‐DPAT) induces an augmentation of Na+,K+‐ATPase activity that results from the recruitment of enzyme molecules to the plasmalemma. Cells expressing the rodent wild‐type Na+,K+‐ATPase α‐subunit had the same basal Na+,K+‐ATPase activity as cells expressing the α‐subunit S11A or S18A mutants, but stimulation of Na+,K+‐ATPase activity was completely abolished in either mutant. 8‐OH‐DPAT treatment of OK cells led to PKCβ‐dependent phosphorylation of the α‐subunit Ser‐11 and Ser‐18 residues, and determination of enzyme activity with the S11A and S18A mutants indicated that both residues are essential for the agonist‐dependent stimulation of Na+,K+‐ATPase activity. When cells were treated with both dopamine and 8‐OH‐DPAT, an activation of Na+,K+‐ATPase was observed at basal intracellular sodium concentration (∼9 mM), and this activation was gradually reduced and became a significant inhibition as the concentration of intracellular sodium gradually increased from 9 to 19 mM. Thus, besides the antagonistic effects of dopamine and 8‐OH‐DPAT, intracellular sodium modulates whether an activation or an inhibition of Na+,K+‐ATPase is produced.

Trafficking of Na-K-ATPase and dopamine receptor molecules induced by changes in intracellular sodium concentration of renal epithelial cells.

Most of the transepithelial transport of sodium in proximal tubules occurs through the coordinated action of the apical sodium/proton exchanger and the basolateral Na-K-ATPase. Hormones that regulate proximal tubule sodium excretion regulate the activities of these proteins. We have previously demonstrated that the level of intracellular sodium concentration modulates the regulation of Na-K-ATPase activity by angiotensin II and dopamine. An increase of a few millimolars in intracellular sodium concentration leads to increased Na-K-ATPase activity without a statistically significant increase in the number of plasma membrane Na-K-ATPase molecules, as determined by cell surface protein biotinylation. Using total internal reflection fluorescence, we detected an increased number of Na-K-ATPase molecules in cytosolic compartments adjacent to the plasma membrane, suggesting that the increased intracellular sodium concentration induces a movement of Na-K-ATPase molecules toward the plasma membrane. While intracellular compartments containing Na-K-ATPase molecules are very close to the plasma membrane, compartments containing type 1 dopamine receptors (D1Rs) are distributed in different parts of the cell cytosol. Fluorescence determinations indicate that an increased intracellular sodium concentration induces the increased colocalization of dopamine receptors with Na-K-ATPase molecules in the region of the plasma membrane. We propose that under in vivo conditions, in response to a sodium load in the lumen of proximal tubules, an increased level of intracellular sodium in epithelial cells is an early event that triggers the cellular response that leads to dopamine inhibition of proximal tubule sodium reabsorption.

Contrary to Rat-Type, Human-Type Na,K-ATPase Is Phosphorylated at the Same Amino Acid by Hormones that Produce Opposite Effects on Enzyme Activity

Renal sodium homeostasis is a major determinant of BP and is regulated by several natriuretic and antinatriuretic hormones. These hormones, acting through intracellular secondary messengers, either activate or inhibit proximal tubule Na,K-ATPase. It was shown previously that phorbol esters and angiotensin II and serotonin induce the phosphorylation of both Ser-11 and Ser-18 of the Na,K-ATPase alpha-subunit. This results in the recruitment of Na,K-ATPase molecules to the plasma membrane and an increased capacity to transport sodium ions. Treatment of the same cells with dopamine leads to phosphorylation of the Na,K-ATPase alpha-subunit Ser-18. The subsequent internalization of Na,K-ATPase molecules results in a reduced capacity to transport sodium ions. These effects are observed in cells that express the rat-type Na,K-ATPase. However, the Na,K-ATPase alpha1-subunit of several species, such as human, pig, and mouse, does not have a Ser-18 in their N-terminal region. Therefore, the possibility exists that, in those species, the Na,K-ATPase is not regulated by the hormones that regulate natriuresis. This study presents evidence that in cells that express the human-type Na,K-ATPase, dopamine inhibits and phorbol esters activate the Na,K-ATPase-mediated transport. These opposite effects are mediated by the phosphorylation of the same amino acid residue, Ser-11 of Na,K-ATPase alpha1, and the presence of alpha1 Ser-18 is not essential for the hormonal regulation of Na,K-ATPase activity in LLCPK1 cells. It was observed that, whereas the regulatory stimulation of Na,K-ATPase is mediated by protein kinase Cbeta, the regulatory inhibition is mediated by protein kinase Czeta. This is similar to what was demonstrated previously in cells that express the rat-type Na,K-ATPase.

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

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.

FRET analysis reveals a critical conformational change within the Na,K-ATPase α1 subunit N-terminus during GPCR-dependent endocytosis

Dopamine is a major regulator of sodium reabsorption in proximal tubule epithelia. It induces the endocytosis of plasma membrane Na,K‐ATPase molecules, and this results in a reduced capacity of the cells to transport sodium. Dopamine induces the phosphorylation of Ser‐18 in the α1‐subunit of Na,K‐ATPase. Fluorescence resonance energy transfer analysis of cells expressing YFP‐α1 and β1‐CFP reveals that treatment of the cells with dopamine increases energy transfer between CFP and YFP. This is consistent with a protein conformational change that results in the N‐terminal end of α1 moving closer to the internal face of the plasma membrane.

G-protein-coupled Receptor-mediated Traffic of Na,K-ATPase to the Plasma Membrane Requires the Binding of Adaptor Protein 1 to a Tyr-255-based Sequence in the α-Subunit

Motion of integral membrane proteins to the plasma membrane in response to G-protein-coupled receptor signals requires selective cargo recognition motifs that bind adaptor protein 1 and clathrin. Angiotensin II, through the activation of AT1 receptors, promotes the recruitment to the plasma membrane of Na,K-ATPase molecules from intracellular compartments. We present evidence to demonstrate that a tyrosine-based sequence (IVVY-255) present within the Na,K-ATPase α1-subunit is involved in the binding of adaptor protein 1. Mutation of Tyr-255 to a phenylalanine residue in the Na,K-ATPase α1-subunit greatly reduces the angiotensin II-dependent activation of Na,K-ATPase, recruitment of Na,K-ATPase molecules to the plasma membrane, and association of adaptor protein 1 with Na,K-ATPase α1-subunit molecules. To determine protein-protein interaction, we used fluorescence resonance energy transfer between fluorophores attached to the Na,K-ATPase α1-subunit and adaptor protein 1. Although angiotensin II activation of AT1 receptors induces a significant increase in the level of fluorescence resonance energy transfer between the two molecules, this effect was blunted in cells expressing the Tyr-255 mutant. Thus, results from different methods and techniques suggest that the Tyr-255-based sequence within the NKA α1-subunit is the site of adaptor protein 1 binding in response to the G-protein-coupled receptor signals produced by angiotensin II binding to AT1 receptors.