title: Functional expression of mutations in the human NaCl cotransporter: evidence for impaired routing mechanisms in Gitelman's syndrome.
abstract: 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.
authors.affiliation: Department of Cell Physiology, University Medical Center Nijmegen, Nijmegen, The Netherlands.
authors.forename: Joke C
authors.initials: JC
authors.lastname: De Jong
authors.forename: Walter A
authors.initials: WA
authors.lastname: Van Der Vliet
authors.forename: Lambertus P W J
authors.initials: LP
authors.lastname: Van Den Heuvel
authors.forename: Peter H G M
authors.initials: PH
authors.lastname: Willems
authors.forename: Nine V A M
authors.initials: NV
authors.lastname: Knoers
authors.forename: René J M
authors.initials: RJ
authors.lastname: Bindels
mesh_terms: D000471:Alkalosis / Q000235:genetics*; D000818:Animals; D002118:Calcium / Q000652:urine*; D002352:Carrier Proteins / Q000235:genetics*; D005260:Female; D006031:Glycosylation; D006801:Humans; D007008:Hypokalemia / Q000235:genetics*; D007150:Immunohistochemistry; D008274:Magnesium / Q000097:blood*; D008788:Metolazone / Q000494:pharmacology; D008970:Molecular Weight; D009154:Mutation; D009865:Oocytes / Q000378:metabolism; D011955:Receptors, Drug; D012964:Sodium / Q000378:metabolism; D049992:Sodium Chloride Symporters; D064486:Solute Carrier Family 12, Member 3; D027981:Symporters; D013577:Syndrome; D014982:Xenopus laevis
chemical_list: D002352:Carrier Proteins; D011955:Receptors, Drug; C578874:SLC12A3 protein, human; D049992:Sodium Chloride Symporters; D064486:Solute Carrier Family 12, Member 3; D027981:Symporters; C064415:thiazide receptor; D012964:Sodium; D008274:Magnesium; D002118:Calcium; D008788:Metolazone