Functional expression of mutations in the human NaCl cotransporter: Evidence for impaired routing mechanisms in gitelman's syndrome

• Published in: Journal of the American Society of Nephrology Vol. 13; no. 6; pp. 1442 - 1448
• Main Authors: DE JONG, Joke C, VAN DER VLIET, Walter A, VAN DEN HEUVEL, Lambertus P. W. J, WILLEMS, Peter H. G. M, KNOERS, Nine V. A. M, BINDELS, René J. M
• Format: Journal Article
• Language: English
• Published: Hagerstown, MD Lippincott Williams & Wilkins 01.06.2002
• Subjects: Alkalosis - genetics; Anesthesia. Intensive care medicine. Transfusions. Cell therapy and gene therapy; Animals; Biological and medical sciences; Calcium - urine; Carrier Proteins - genetics; Emergency and intensive care: metabolism and nutrition disorders. Enteral and parenteral nutrition; Female; Glycosylation; Humans; Hypokalemia - genetics; Immunohistochemistry; Intensive care medicine; Magnesium - blood; Medical sciences; Metolazone - pharmacology; Molecular Weight; Mutation; Oocytes - metabolism; Receptors, Drug; Sodium - metabolism; Sodium Chloride Symporters; Solute Carrier Family 12, Member 3; Symporters; Syndrome; Xenopus laevis; Human; Pathogenesis; Metabolic alkalosis; Biological transport; Acid base balance disorder; Inorganic element; Genetic disease; Metabolic disorder; Gene; Sodium; Autosomal character; Chlorides; Hypocalciuria; Recessive character; Hypokaliemia; Gitelman's syndrome; Mutation; Hypomagnesemia
• ISSN: 1046-6673; 1533-3450
• DOI: 10.1097/01.ASN.0000017904.77985.03

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.