title: NKCC2 activity is inhibited by the Bartter's syndrome type 5 gain-of-function CaR-A843E mutant in renal cells.
abstract: BACKGROUND
  The gain-of-function A843E mutation of the calcium sensing receptor (CaR) causes Bartter syndrome type 5. Patients carrying this CaR variant show a remarkably reduced renal NaCl reabsorption in the thick ascending limb (TAL) of Henle's loop resulting in renal loss of NaCl in the absence of mutations in renal Na(+) and Cl(-) ion transporters. The molecular mechanisms underlying this clinical phenotype are incompletely understood. We investigated, in human embryonic kidney 293 (HEK 293) cells and porcine kidney epithelial (LLC-PK1) cells, the functional cross-talk of CaR-A843E with the Na(+):K(+):2Cl(-) co-transporter, NKCC2, which provides NaCl reabsorption in the TAL.
  
  
  RESULTS
  The expression of the CaR mutant did not alter the apical localisation of NKCC2 in LLC-PK1 cells. However, the steady-state NKCC2 phosphorylation and activity were decreased in cells transfected with CaR-A843E compared with the control wild-type CaR (CaR WT)-transfected cells. Of note, low-Cl(-)-dependent NKCC2 activation was also strongly inhibited upon the expression of CaR-A843E mutant. The use of either P450 ω-hydroxylase (CYP4)- or phospholipase A2 (PLA2)-blockers suggests that this effect is likely mediated by arachidonic acid (AA) metabolites.
  
  
  CONCLUSIONS
  The data suggested that the activated CaR affects intracellular pathways modulating NKCC2 activity rather than NKCC2 intracellular trafficking in renal cells, and throw further light on the pathological role played by active CaR mutants in Bartter syndrome type 5.
authors: Department of Biosciences, Biotechnologies, and Biopharmaceutics, University of Bari, 70125 Bari, Italy; Department of Sciences, University of Basilicata, 85100 Potenza, Italy.
authors: Monica
authors: M
authors: Carmosino
authors: Andrea
authors: A
authors: Gerbino
authors: Geoffrey N
authors: GN
authors: Hendy
authors: Silvia
authors: S
authors: Torretta
authors: Federica
authors: F
authors: Rizzo
authors: Lucantonio
authors: L
authors: Debellis
authors: Giuseppe
authors: G
authors: Procino
authors: Maria
authors: M
authors: Svelto
mesh_terms: D000818:Animals; D001477:Bartter Syndrome / Q000235:genetics* / Q000378:metabolism*; D002460:Cell Line; D006801:Humans; D009154:Mutation; D021381:Protein Transport / Q000502:physiology; D044169:Receptors, Calcium-Sensing / Q000235:genetics*; D064466:Solute Carrier Family 12, Member 1 / Q000378:metabolism*; D013552:Swine; D014162:Transfection
chemical_list: D044169:Receptors, Calcium-Sensing; D064466:Solute Carrier Family 12, Member 1
keywords: Calcium homoeostasis/signalling; G-protein-coupled receptors; Intracellular signalling; Kidney; Lipid mediator