Functional expression of mutations in the human NaCl cotransporter: evidence for impaired routing mechanisms in Gitelman's syndrome.
SourceJournal of the American Society of Nephrology, 13, 6, (2002), pp. 1442-8
Article / Letter to editor
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Paediatrics - OUD tm 2017
Journal of the American Society of Nephrology
SubjectElucidation of hereditary disorders and their molecular diagnosis; Regulation of salt and water reabsorption in the renal collecting duct; Signal Transduction and Ion Transport; Disturbances in biochemical and functional development of the kidney during childhood.; Opheldering van erfelijke ziekten en hun moleculaire diagnostiek; Regulatie water en zouttransport in de verzamelbuis van de nier; Signaaltransductie en ionentransport; Stoornissen in de biochemische en functionele ontwikkeling van de nier op kinderleeftijd
Gitelman's syndrome is an autosomal recessive renal tubular disorder characterized by hypokalemic metabolic alkalosis, hypomagnesemia, and hypocalciuria. This disorder results from mutations in the thiazide-sensitive NaCl cotransporter (NCC). To elucidate the functional implications of mutations associated with this disorder, metolazone-sensitive (22)Na(+) uptake, subcellular localization, and glycosidase-sensitive glycosylation of human NCC (hNCC) were determined in Xenopus laevis oocytes expressing FLAG-tagged wild-type or mutant hNCC. Injection of 10 ng of FLAG-tagged hNCC cRNA resulted in metolazone-sensitive (22)Na(+) uptake of 3.4 +/- 0.2 nmol Na(+)/oocyte per 2 h. Immunocytochemical analysis revealed sharp immunopositive staining at the plasma membrane. In agreement with this finding, a broad endoglycosidase H-insensitive band of 130 to 140 kD was present in Western blots of total membranes. The plasma membrane localization of this complex-glycosylated protein was confirmed by immunoblotting of purified plasma membranes. The mutants could be divided into two distinct classes. Class I mutants (G439S, T649R, and G741R) exhibited no significant metolazone-sensitive (22)Na(+) uptake. Immunopositive staining was present in a diffuse band just below the plasma membrane. This endoplasmic reticulum and/or pre-Golgi complex localization was further suggested by the complete absence of the endoglycosidase H-insensitive band. Class II mutants (L215P, F536L, R955Q, G980R, and C985Y) demonstrated significant metolazone-sensitive (22)Na(+) uptake, although uptake was significantly lower than that obtained with wild-type hNCC. The latter mutants could be detected at and below the oocyte plasma membrane, and immunoblotting revealed the characteristic complex-glycosylated bands. In conclusion, this study substantiates NCC processing defects as the underlying pathogenic mechanism in Gitelman's syndrome.
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