Article: article from journal or magazin.
Functional assessment of sodium chloride cotransporter NCC mutants in polarized mammalian epithelial cells.
American journal of physiology. Renal physiology
Publication types: Comparative Study ; Journal Article
Publication Status: ppublish
Publication Status: ppublish
The thiazide-sensitive sodium chloride cotransporter NCC is important for maintaining serum sodium (Na(+)) and, indirectly, serum potassium (K(+)) levels. Functional studies on NCC have used cell lines with native NCC expression, transiently transfected nonpolarized cell lines, or Xenopus laevis oocytes. Here, we developed the use of polarized Madin-Darby canine kidney type I (MDCKI) mammalian epithelial cell lines with tetracycline-inducible human NCC expression to study NCC activity and membrane abundance in the same system. In radiotracer assays, induced cells grown on filters had robust thiazide-sensitive and chloride dependent sodium-22 ((22)Na) uptake from the apical side. To minimize cost and maximize throughput, assays were modified to use cells grown on plastic. On plastic, cells had similar thiazide-sensitive (22)Na uptakes that increased following preincubation of cells in chloride-free solutions. NCC was detected in the plasma membrane, and both membrane abundance and phosphorylation of NCC were increased by incubation in chloride-free solutions. Furthermore, in cells exposed for 15 min to low or high extracellular K(+), the levels of phosphorylated NCC increased and decreased, respectively. To demonstrate that the system allows rapid and systematic assessment of mutated NCC, three phosphorylation sites in NCC were mutated, and NCC activity was examined. (22)Na fluxes in phosphorylation-deficient mutants were reduced to baseline levels, whereas phosphorylation-mimicking mutants were constitutively active, even without chloride-free stimulation. In conclusion, this system allows the activity, cellular localization, and abundance of wild-type or mutant NCC to be examined in the same polarized mammalian expression system in a rapid, easy, and low-cost fashion.
Animals, Cell Culture Techniques, Cell Polarity, Chlorides/metabolism, Dogs, Dose-Response Relationship, Drug, Epithelial Cells/drug effects, Epithelial Cells/metabolism, Genotype, High-Throughput Screening Assays, Kinetics, Madin Darby Canine Kidney Cells, Mutation, Phenotype, Phosphorylation, Potassium/metabolism, Protein Processing, Post-Translational, Sodium/metabolism, Sodium Chloride Symporter Inhibitors/pharmacology, Solute Carrier Family 12, Member 3/drug effects, Solute Carrier Family 12, Member 3/genetics, Solute Carrier Family 12, Member 3/metabolism, Transfection, Na+/Cl− cotransporter, Slc12a3, cotransporter, phosphorylation, sodium flux assay, thiazide-sensitive cotransporter
Web of science
Last modification date