Mutations in the Human Na-K-2Cl Cotransporter (NKCC2) Identified in Bartter Syndrome Type I Consistently Result in Nonfunctional Transporters
Copyright policy )Mutations in the Human Na-K-2Cl Cotransporter (NKCC2) Identified in Bartter Syndrome Type I Consistently Result in Nonfunctional Transporters
Bartter syndrome (BS) is a heterogeneous renal tubular disorder affecting Na-K-Cl reabsorption in the thick ascending limb of Henle's loop. BS type I patients typically present with profound hypokalemia and metabolic alkalosis. The main goal of the present study was to elucidate the functional implications of six homozygous mutations (G193R, A267S, G319R, A508T, del526N, and Y998X) in the bumetanide-sensitive Na-K-2Cl cotransporter (hNKCC2) identified in patients diagnosed with BS type I. To this end, capped RNA (cRNA) of FLAG-tagged hNKCC2 and the corresponding mutants was injected in Xenopus laevis oocytes and transporter activity was measured after 72 h by means of a bumetanide-sensitive (22)Na(+) uptake assay at 30 degrees C. Injection of 25 ng of hNKCC2 cRNA resulted in bumetanide-sensitive (22)Na(+) uptake of 2.5 +/- 0.5 nmol/oocyte per 30 min. Injection of 25 ng of mutant cRNA yielded no significant bumetanide-sensitive (22)Na(+) uptake. Expression of wild-type and mutant transporters was confirmed by immunoblotting, showing significantly less mutant protein compared with wild-type at the same cRNA injection levels. However, when the wild-type cRNA injection level was reduced to obtain a protein expression level equal to that of the mutants, the wild-type still exhibited a significant bumetanide-sensitive (22)Na(+) uptake. Immunocytochemical analysis showed immunopositive staining of hNKCC2 at the plasma membrane for wild-type and all studied mutants. In conclusion, mutations in hNKCC2 identified in type I BS patients, when expressed in Xenopus oocytes, result in a low expression of normally routed but functionally impaired transporters. These results are in line with the hypothesis that the mutations in hNKCC2 are the underlying cause of the clinical abnormalities seen in patients with type I BS.
- Radboud University Nijmegen Netherlands
- Radboud University Nijmegen Medical Centre Netherlands
Glycoside Hydrolases, Sodium-Potassium-Chloride Symporters, Immunoblotting, Sodium, Molecular Conformation, Bartter Syndrome, UMCN 5.4: Renal disorders, Immunohistochemistry, UMCN 5.1: Genetic defects of metabolism, Xenopus laevis, UMCN 5.3: Cellular energy metabolism, Mutation, Oocytes, Animals, Humans, Protein Processing, Post-Translational, Bumetanide, Solute Carrier Family 12, Member 1
Glycoside Hydrolases, Sodium-Potassium-Chloride Symporters, Immunoblotting, Sodium, Molecular Conformation, Bartter Syndrome, UMCN 5.4: Renal disorders, Immunohistochemistry, UMCN 5.1: Genetic defects of metabolism, Xenopus laevis, UMCN 5.3: Cellular energy metabolism, Mutation, Oocytes, Animals, Humans, Protein Processing, Post-Translational, Bumetanide, Solute Carrier Family 12, Member 1
citations This is an alternative to the "Influence" indicator, which also reflects the overall/total impact of an article in the research community at large, based on the underlying citation network (diachronically).65 popularity This indicator reflects the "current" impact/attention (the "hype") of an article in the research community at large, based on the underlying citation network.Top 10% influence This indicator reflects the overall/total impact of an article in the research community at large, based on the underlying citation network (diachronically).Top 10% impulse This indicator reflects the initial momentum of an article directly after its publication, based on the underlying citation network.Top 10%
Citations provided by BIP!Popularity provided by BIP!