Publication

The structural unit of the thiazide-sensitive NaCl cotransporter is a homodimer

de Jong, J. C., Willems, P. H. G. M., Mooren, F. J. M., van den Heuvel, L. P. W. J., Knoers, N. V. A. M. & Bindels, R. J. M., 4-Jul-2003, In : The Journal of Biological Chemistry. 278, 27, p. 24302-7 6 p.

Research output: Contribution to journalArticleAcademicpeer-review

APA

de Jong, J. C., Willems, P. H. G. M., Mooren, F. J. M., van den Heuvel, L. P. W. J., Knoers, N. V. A. M., & Bindels, R. J. M. (2003). The structural unit of the thiazide-sensitive NaCl cotransporter is a homodimer. The Journal of Biological Chemistry, 278(27), 24302-7. https://doi.org/10.1074/jbc.M303101200

Author

de Jong, Joke C ; Willems, Peter H G M ; Mooren, Fieke J M ; van den Heuvel, Lambertus P W J ; Knoers, Nine V A M ; Bindels, René J M. / The structural unit of the thiazide-sensitive NaCl cotransporter is a homodimer. In: The Journal of Biological Chemistry. 2003 ; Vol. 278, No. 27. pp. 24302-7.

Harvard

de Jong, JC, Willems, PHGM, Mooren, FJM, van den Heuvel, LPWJ, Knoers, NVAM & Bindels, RJM 2003, 'The structural unit of the thiazide-sensitive NaCl cotransporter is a homodimer', The Journal of Biological Chemistry, vol. 278, no. 27, pp. 24302-7. https://doi.org/10.1074/jbc.M303101200

Standard

The structural unit of the thiazide-sensitive NaCl cotransporter is a homodimer. / de Jong, Joke C; Willems, Peter H G M; Mooren, Fieke J M; van den Heuvel, Lambertus P W J; Knoers, Nine V A M; Bindels, René J M.

In: The Journal of Biological Chemistry, Vol. 278, No. 27, 04.07.2003, p. 24302-7.

Research output: Contribution to journalArticleAcademicpeer-review

Vancouver

de Jong JC, Willems PHGM, Mooren FJM, van den Heuvel LPWJ, Knoers NVAM, Bindels RJM. The structural unit of the thiazide-sensitive NaCl cotransporter is a homodimer. The Journal of Biological Chemistry. 2003 Jul 4;278(27):24302-7. https://doi.org/10.1074/jbc.M303101200


BibTeX

@article{37c03594066f4cba8af774e76093606f,
title = "The structural unit of the thiazide-sensitive NaCl cotransporter is a homodimer",
abstract = "The thiazide-sensitive NaCl cotransporter (NCC) is responsible for the reabsorption of 5{\%} of the filtered load of NaCl in the kidney. Mutations in NCC cause Gitelman syndrome. To gain insight into its regulation, detailed information on the structural composition of its functional unit is essential. Western blot analysis of total membranes of Xenopus laevis oocytes heterologously expressing FLAG-tagged NCC revealed the presence of both complex-(140-kDa) and core (100-kDa)-glycosylated monomers and a broad band of high molecular mass (250-350-kDa) complexes. Chemical cross-linking with dithiobispropionimidate eliminated the low molecular weight bands and increased the intensity of the high molecular weight bands, indicating that NCC is present in multimeric complexes. Co-expression of HA- and FLAG-tagged NCC followed by co-immunoprecipitation demonstrated that these multimers contained at least two complex-glycosylated NCC proteins. The dimeric nature of the multimers was further substantiated by sucrose gradient centrifugation yielding a peak of approximately 310 kDa. A concatameric construct of two NCC polyproteins exhibited significant 22Na+ uptake, indicating that the transporter is functional as a homodimer. A concatamer of partially retarded G980R- and wild type (wt)-NCC displayed normal Na+ transport. This demonstrates that G980R-NCC, provided that it reaches the surface, is fully active and that wt-NCC is dominant in its association with this mutant. Conversely a concatamer of fully retarded G741R- and wt-NCC did not reach the cell surface, showing that wt-NCC is recessive in its association with this mutant. Oocytes co-expressing G741R- and wt-NCC did not show G741R staining at the plasma membrane, whereas Na+ transport was normal, indicating that wt-NCC dimerizes preferentially with itself. The results are discussed in relation to the recessive nature of NCC mutants in Gitelman syndrome.",
keywords = "Animals, Dimerization, Humans, Hypokalemia/etiology, Mutation, Sodium-Potassium-Chloride Symporters/chemistry, Thiazines, Xenopus",
author = "{de Jong}, {Joke C} and Willems, {Peter H G M} and Mooren, {Fieke J M} and {van den Heuvel}, {Lambertus P W J} and Knoers, {Nine V A M} and Bindels, {Ren{\'e} J M}",
year = "2003",
month = "7",
day = "4",
doi = "10.1074/jbc.M303101200",
language = "English",
volume = "278",
pages = "24302--7",
journal = "The Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC",
number = "27",

}

RIS

TY - JOUR

T1 - The structural unit of the thiazide-sensitive NaCl cotransporter is a homodimer

AU - de Jong, Joke C

AU - Willems, Peter H G M

AU - Mooren, Fieke J M

AU - van den Heuvel, Lambertus P W J

AU - Knoers, Nine V A M

AU - Bindels, René J M

PY - 2003/7/4

Y1 - 2003/7/4

N2 - The thiazide-sensitive NaCl cotransporter (NCC) is responsible for the reabsorption of 5% of the filtered load of NaCl in the kidney. Mutations in NCC cause Gitelman syndrome. To gain insight into its regulation, detailed information on the structural composition of its functional unit is essential. Western blot analysis of total membranes of Xenopus laevis oocytes heterologously expressing FLAG-tagged NCC revealed the presence of both complex-(140-kDa) and core (100-kDa)-glycosylated monomers and a broad band of high molecular mass (250-350-kDa) complexes. Chemical cross-linking with dithiobispropionimidate eliminated the low molecular weight bands and increased the intensity of the high molecular weight bands, indicating that NCC is present in multimeric complexes. Co-expression of HA- and FLAG-tagged NCC followed by co-immunoprecipitation demonstrated that these multimers contained at least two complex-glycosylated NCC proteins. The dimeric nature of the multimers was further substantiated by sucrose gradient centrifugation yielding a peak of approximately 310 kDa. A concatameric construct of two NCC polyproteins exhibited significant 22Na+ uptake, indicating that the transporter is functional as a homodimer. A concatamer of partially retarded G980R- and wild type (wt)-NCC displayed normal Na+ transport. This demonstrates that G980R-NCC, provided that it reaches the surface, is fully active and that wt-NCC is dominant in its association with this mutant. Conversely a concatamer of fully retarded G741R- and wt-NCC did not reach the cell surface, showing that wt-NCC is recessive in its association with this mutant. Oocytes co-expressing G741R- and wt-NCC did not show G741R staining at the plasma membrane, whereas Na+ transport was normal, indicating that wt-NCC dimerizes preferentially with itself. The results are discussed in relation to the recessive nature of NCC mutants in Gitelman syndrome.

AB - The thiazide-sensitive NaCl cotransporter (NCC) is responsible for the reabsorption of 5% of the filtered load of NaCl in the kidney. Mutations in NCC cause Gitelman syndrome. To gain insight into its regulation, detailed information on the structural composition of its functional unit is essential. Western blot analysis of total membranes of Xenopus laevis oocytes heterologously expressing FLAG-tagged NCC revealed the presence of both complex-(140-kDa) and core (100-kDa)-glycosylated monomers and a broad band of high molecular mass (250-350-kDa) complexes. Chemical cross-linking with dithiobispropionimidate eliminated the low molecular weight bands and increased the intensity of the high molecular weight bands, indicating that NCC is present in multimeric complexes. Co-expression of HA- and FLAG-tagged NCC followed by co-immunoprecipitation demonstrated that these multimers contained at least two complex-glycosylated NCC proteins. The dimeric nature of the multimers was further substantiated by sucrose gradient centrifugation yielding a peak of approximately 310 kDa. A concatameric construct of two NCC polyproteins exhibited significant 22Na+ uptake, indicating that the transporter is functional as a homodimer. A concatamer of partially retarded G980R- and wild type (wt)-NCC displayed normal Na+ transport. This demonstrates that G980R-NCC, provided that it reaches the surface, is fully active and that wt-NCC is dominant in its association with this mutant. Conversely a concatamer of fully retarded G741R- and wt-NCC did not reach the cell surface, showing that wt-NCC is recessive in its association with this mutant. Oocytes co-expressing G741R- and wt-NCC did not show G741R staining at the plasma membrane, whereas Na+ transport was normal, indicating that wt-NCC dimerizes preferentially with itself. The results are discussed in relation to the recessive nature of NCC mutants in Gitelman syndrome.

KW - Animals

KW - Dimerization

KW - Humans

KW - Hypokalemia/etiology

KW - Mutation

KW - Sodium-Potassium-Chloride Symporters/chemistry

KW - Thiazines

KW - Xenopus

U2 - 10.1074/jbc.M303101200

DO - 10.1074/jbc.M303101200

M3 - Article

VL - 278

SP - 24302

EP - 24307

JO - The Journal of Biological Chemistry

JF - The Journal of Biological Chemistry

SN - 0021-9258

IS - 27

ER -

ID: 92781459