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The Structural Unit of the Thiazide-sensitive NaCl Cotransporter Is a Homodimer
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....
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Published in: | The Journal of biological chemistry 2003-07, Vol.278 (27), p.24302-24307 |
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Main Authors: | , , , , , |
Format: | Article |
Language: | English |
Subjects: | |
Citations: | Items that this one cites Items that cite this one |
Online Access: | Get full text |
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Summary: | 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 (250â350-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 â¼310 kDa. A concatameric construct of two NCC polyproteins
exhibited significant 22 Na + 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. |
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ISSN: | 0021-9258 1083-351X |
DOI: | 10.1074/jbc.M303101200 |