Blocking the salt-inducible kinase 1 network prevents the increases in cell sodium transport caused by a hypertension-linked mutation in human alpha-adducin.

Objectives: Because a newly described salt-inducible kinase 1 (SIK1) network is responsible for increases in active cell sodium transport in response to elevated intracellular sodium, we hypothesized that this network could mediate the effects of the mutant (hypertensive) form of alpha-adducin on Na,K-ATPase activity.

Methods: Studies were performed in normotensive and hypertensive Milan rats and in a cell line of proximal tubule origin expressing transiently variants of alpha-adducin (human G460W/S586C; rat F316Y) that are associated with elevated blood pressure and result in increased Na,K-ATPase activity. Na,K-ATPase activity was determined as ouabain-sensitive rubidium transport.

SIK1 is part of a cell sodium-sensing network that regulates active sodium transport through a calcium-dependent process.

In mammalian cells, active sodium transport and its derived functions (e.g., plasma membrane potential) are dictated by the activity of the Na(+),K(+)-ATPase (NK), whose regulation is essential for maintaining cell volume and composition, as well as other vital cell functions.

Phosphorylation of adaptor protein-2 mu2 is essential for Na+,K+-ATPase endocytosis in response to either G protein-coupled receptor or reactive oxygen species.

Activation of G protein-coupled receptor by dopamine and hypoxia-generated reactive oxygen species promote Na+,K+-ATPase endocytosis. This effect is clathrin dependent and involves the activation of protein kinase C (PKC)-zeta and phosphorylation of the Na+,K+-ATPase alpha-subunit. Because the incorporation of cargo into clathrin vesicles requires association with adaptor proteins, we studied whether phosphorylation of adaptor protein (AP)-2 plays a role in its binding to the Na+,K+-ATPase alpha-subunit and thereby in its endocytosis.

The 14-3-3 protein translates the NA+,K+-ATPase {alpha}1-subunit phosphorylation signal into binding and activation of phosphoinositide 3-kinase during endocytosis.

Clathrin-dependent endocytosis of Na(+),K(+)-ATPase molecules in response to G protein-coupled receptor signals is triggered by phosphorylation of the alpha-subunit and the binding of phosphoinositide 3-kinase. In this study, we describe a molecular mechanism linking phosphorylation of Na(+),K(+)-ATPase alpha-subunit to binding and activation of phosphoinositide 3-kinase. Co-immunoprecipitation studies, as well as experiments using confocal microscopy, revealed that dopamine favored the association of 14-3-3 protein with the basolateral plasma membrane and its co-localization with the Na(+),K(+)-ATPase alpha-subunit.

Hypertension-linked mutation in the adducin alpha-subunit leads to higher AP2-mu2 phosphorylation and impaired Na+,K+-ATPase trafficking in response to GPCR signals and intracellular sodium.

Alpha-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.

Isoform-specific regulation of Na+,K+-ATPase endocytosis and recruitment to the plasma membrane.

The Na(+),K(+)-ATPase traffics between the plasma membrane and intracellular compartments in response to acute changes in membrane receptor activation. These effects are accomplished by a time-dependent interaction of the Na(+),K(+)-ATPase alpha-subunit with specific intracellular signaling molecules either at the plasma membrane (endocytosis) or at the endosome's membranes (recruitment). Most of these studies have been performed in rat renal epithelial cells in which only the alpha(1) isoenzyme is present.

Relevance of dopamine signals anchoring dynamin-2 to the plasma membrane during Na+,K+-ATPase endocytosis.

Clathrin-dependent endocytosis of Na(+),K(+)-ATPase in response to dopamine regulates its catalytic activity in intact cells. Because fission of clathrin-coated pits requires dynamin, we examined the mechanisms by which dopamine receptor signals promote dynamin-2 recruitment and assembly at the site of Na(+),K(+)-ATPase endocytosis. Western blotting revealed that dopamine increased the association of dynamin-2 with the plasma membrane and with phosphatidylinositol 3-kinase.

Dopamine-induced exocytosis of Na,K-ATPase is dependent on activation of protein kinase C-epsilon and -delta.

The purpose of this study was to define mechanisms by which dopamine (DA) regulates the Na,K-ATPase in alveolar epithelial type 2 (AT2) cells. The Na,K-ATPase activity increased by twofold in cells incubated with either 1 microM DA or a dopaminergic D(1) agonist, fenoldopam, but not with the dopaminergic D(2) agonist quinpirole. The increase in activity paralleled an increase in Na,K-ATPase alpha1 and beta1 protein abundance in the basolateral membrane (BLM) of AT2 cells.

Tyrosine 537 within the Na+,K+-ATPase alpha-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 alpha(1)-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 alpha(1)-subunit. We identified a conserved sequence (Y(537)LEL) within the alpha(1)-subunit that represents an AP-2 binding site.