cAMP-dependent activation of the renal-specific Na+-K+-2Cl−cotransporter is mediated by regulation of cotransporter trafficking

Meade, Patricia; Hoover, Robert S.; Plata, Consuelo; Vázquez, Norma; Bobadilla, Norma A.; Gamba, Gerardo; Hebert, Steven C.

doi:10.1152/ajprenal.00421.2002

Cited by 56 publications

(54 citation statements)

References 36 publications

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“…This effect is attributed, at least in part, to an increased NKCC2 translocation to the apical membrane (10,11). Interestingly, functional expression in X. laevis oocytes showed that the C-terminal truncated isoform of NKCC2 (S-NKCC2) reduced L-NKCC2 activity by preventing arrival of the co-transporter to the plasma membrane, an effect prevented by cAMP (71,77). Most importantly, these studies demonstrated that in the presence of S-NKCC2, but not in its absence, L-NKCC2 surface level is increased by cAMP-dependent protein kinase activation.…”

Section: Discussionmentioning

confidence: 99%

NKCC2 Surface Expression in Mammalian Cells

Benziane

Demaretz

Defontaine

et al. 2007

Journal of Biological Chemistry

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Apical bumetanide-sensitive Na؉ -K ؉ -2Cl ؊ co-transporter, termed NKCC2, is the major salt transport pathway in kidney thick ascending limb. NKCC2 surface expression is subject to regulation by intracellular protein trafficking. However, the protein partners involved in the intracellular trafficking of NKCC2 remain unknown. Moreover, studies aimed at understanding the post-translational regulation of NKCC2 have been hampered by the difficulty to express NKCC2 protein in mammalian cells. Here we were able to express NKCC2 protein in renal epithelial cells by tagging its N-terminal domain. To gain insights into the regulation of NKCC2 trafficking, we screened for interaction partners of NKCC2 with the yeast two-hybrid system, using the C-terminal tail of NKCC2 as bait. Aldolase B was identified as a dominant and novel interacting protein. Real time PCR on renal microdissected tubules demonstrated the expression of aldolase B in the thick ascending limb. Co-immunoprecipitation and co-immunolocalization experiments confirmed NKCC2-aldolase interaction in renal cells. Biotinylation assays showed that aldolase co-expression reduces NKCC2 surface expression. In the presence of aldolase substrate, fructose 1,6-bisphosphate, aldolase binding was disrupted, and aldolase co-expression had no further effect on the cell surface level of NKCC2. Finally, functional studies demonstrated that aldolase-induced down-regulation of NKCC2 at the plasma membrane was associated with a decrease in its transport activity. In summary, we identified aldolase B as a novel NKCC2 binding partner that plays a key role in the modulation of NKCC2 surface expression, thereby revealing a new regulatory mechanism governing the co-transporter intracellular trafficking. Furthermore, NKCC2 protein expression in mammalian cells and its regulation by protein-protein interactions, described here, may open new and important avenues in studying the cell biology and post-transcriptional regulation of the co-transporter.

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Section: Discussionmentioning

confidence: 99%

NKCC2 Surface Expression in Mammalian Cells

Benziane

Demaretz

Defontaine

et al. 2007

Journal of Biological Chemistry

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“…Transepithelial Mg 2+ reabsorption is passive diffusion according to the positive luminal transepithelial potential (Mandon et al, 1993). The voltage in the lumen of the TAL is determined by the rate of the Na (Kiroytcheva et al, 1999;Meade et al, 2003). PKA inhibitors might indirectly block transepithelial Mg 2+ transport mediated by the inhibition of the Na + -K + -Cl -cotransporter or Na + /K + -ATPase.…”

Section: Discussionmentioning

confidence: 99%

Phosphorylation of paracellin-1 at Ser217 by protein kinase A is essential for localization in tight junctions

Ikari

Matsumoto

Hishida

et al. 2006

Journal of Cell Science

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Although paracellin-1 (PCLN-1) is known to have a crucial role in the control of Mg2+ reabsorption in the kidney, the molecular pathways involved in the regulation of PCLN-1 have not been clarified. We used FLAG-tagged PCLN-1 to investigate these pathways further, and found that PCLN-1 is phosphorylated at Ser217 by protein kinase A (PKA) under physiological conditions in Madin-Darby canine kidney (MDCK) cells. PCLN-1 expression decreased Na+ permeability, resulting in a decrease in the transepithelial electrical resistance (TER). By contrast, PCLN-1 enhanced transepithelial Mg2+ transport. PKA inhibitors, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H-89) and myristoylated protein kinase A inhibitor 14-22 amide PKI, and an adenylate cyclase inhibitor, 2′,5′-dideoxy adenosine (DDA), reduced the phosphoserine level of PCLN-1. The inhibitory effect of DDA was rescued by 8-bromoadenosine-3′,5′-cyclic monophosphate (8-Br-cAMP). PKA and adenylate cyclase inhibitors decreased transepithelial Mg2+ transport and TER. Dephosphorylated PCLN-1 moved from detergent-insoluble to soluble fractions and was dissociated from ZO-1. A fusion protein of PCLN-1 with glutathione-S-transferase revealed that Ser217 was phosphorylated by PKA. Phosphorylated PCLN-1 was localized in the tight junction (TJ) along with ZO-1, whereas dephosphorylated PCLN-1 and the S217A mutant were translocated into the lysosome. The degradation of dephosphorylated PCLN-1 and S217A mutant was inhibited by chloroquine, a specific lysosome inhibitor. Thus, the PKA-dependent phosphorylation of Ser217 in PCLN-1 is essential for its localization in the TJ and transepithelial Mg2+ transport.

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“…Thus Plata et al (325) concluded that the carboxy-terminal truncated isoform of SLC12A1 gene in mouse exerts a dominant-negative function on ion transport by the Na ϩ -K ϩ -2Cl Ϫ cotransporter, a property that is reversed by cAMP. More recently, using a confocal microcopy strategy, in BSC1-L and BSC1-S proteins in which EGFP was attached in frame to the amino-terminal domain, Meade et al (269) observed that the mechanism by which BSC1-S reduced Na ϩ -K ϩ -2Cl Ϫ cotransporter activity is by preventing ar- (324). † Expressed as K m (144).…”

Section: B Apical Bumetanide-sensitive Namentioning

confidence: 99%

Molecular Physiology and Pathophysiology of Electroneutral Cation-Chloride Cotransporters

Gamba

2005

Physiological Reviews

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754

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Electroneutral cation-Cl−cotransporters compose a family of solute carriers in which cation (Na+or K+) movement through the plasma membrane is always accompanied by Cl−in a 1:1 stoichiometry. Seven well-characterized members include one gene encoding the thiazide-sensitive Na+−Cl−cotransporter, two genes encoding loop diuretic-sensitive Na+−K+−2Cl−cotransporters, and four genes encoding K+−Cl−cotransporters. These membrane proteins are involved in several physiological activities including transepithelial ion absorption and secretion, cell volume regulation, and setting intracellular Cl−concentration below or above its electrochemical potential equilibrium. In addition, members of this family play an important role in cardiovascular and neuronal pharmacology and pathophysiology. Some of these cotransporters serve as targets for loop diuretics and thiazide-type diuretics, which are among the most commonly prescribed drugs in the world, and inactivating mutations of three members of the family cause inherited diseases such as Bartter's, Gitelman's, and Anderman's diseases. Major advances have been made in the past decade as consequences of molecular identification of all members in this family. This work is a comprehensive review of the knowledge that has evolved in this area and includes molecular biology of each gene, functional properties of identified cotransporters, structure-function relationships, and physiological and pathophysiological roles of each cotransporter.

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cAMP-dependent activation of the renal-specific Na⁺-K⁺-2Cl⁻cotransporter is mediated by regulation of cotransporter trafficking

Cited by 56 publications

References 36 publications

NKCC2 Surface Expression in Mammalian Cells

NKCC2 Surface Expression in Mammalian Cells

Phosphorylation of paracellin-1 at Ser217 by protein kinase A is essential for localization in tight junctions

Molecular Physiology and Pathophysiology of Electroneutral Cation-Chloride Cotransporters

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