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

被引:70
作者
de Jong, JC
Willems, PHGM
Mooren, FJM
van den Heuvel, LPWJ
Knoers, NVAM
Bindels, RJM
机构
[1] Univ Nijmegen, Med Ctr, Dept Cell Physiol, NL-6500 HB Nijmegen, Netherlands
[2] Univ Nijmegen, Med Ctr, Dept Biochem, NL-6500 HB Nijmegen, Netherlands
[3] Univ Nijmegen, Med Ctr, Dept Pediat, NL-6500 HB Nijmegen, Netherlands
[4] Univ Nijmegen, Med Ctr, Dept Human Genet, NL-6500 HB Nijmegen, Netherlands
关键词
D O I
10.1074/jbc.M303101200
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 similar to310 kDa. A concatameric construct of two NCC polyproteins exhibited significant Na-22(+) 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.
引用
收藏
页码:24302 / 24307
页数:6
相关论文
共 29 条
[1]   A dominant negative mutant of the KCC1K-Cl cotransporter - Both N- and C-terminal cytoplasmic domains are required for K-Cl cotransport activity [J].
Casula, S ;
Shmukler, BE ;
Wilhelm, S ;
Stuart-Tilley, AK ;
Su, WF ;
Chernova, MN ;
Brugnara, C ;
Alper, SL .
JOURNAL OF BIOLOGICAL CHEMISTRY, 2001, 276 (45) :41870-41878
[2]   Functional expression of mutations in the human NaCl cotransporter: Evidence for impaired routing mechanisms in Gitelman's syndrome [J].
De Jong, JC ;
Van der Vliet, WA ;
Van den Heuvel, LPWJ ;
Willems, PHGM ;
Knoers, NVAM ;
Bindels, RJM .
JOURNAL OF THE AMERICAN SOCIETY OF NEPHROLOGY, 2002, 13 (06) :1442-1448
[3]   The epithelial sodium channel (ENaC) is intracellularly located as a tetramer [J].
Dijkink, L ;
Hartog, A ;
van Os, CH ;
Bindels, RJM .
PFLUGERS ARCHIV-EUROPEAN JOURNAL OF PHYSIOLOGY, 2002, 444 (04) :549-555
[4]  
Ellison DH, 2003, J AM SOC NEPHROL, V14, P538, DOI 10.1681/ASN.V142538
[5]   THIAZIDE-SENSITIVE SODIUM-CHLORIDE COTRANSPORT IN EARLY DISTAL TUBULE [J].
ELLISON, DH ;
VELAZQUEZ, H ;
WRIGHT, FS .
AMERICAN JOURNAL OF PHYSIOLOGY, 1987, 253 (03) :F546-F554
[6]  
FAFOURNOUX P, 1994, J BIOL CHEM, V269, P2589
[7]   The heterotetrameric architecture of the epithelial sodium channel (ENaC) [J].
Firsov, D ;
Gautschi, I ;
Merillat, AM ;
Rossier, BC ;
Schild, L .
EMBO JOURNAL, 1998, 17 (02) :344-352
[8]   PRIMARY STRUCTURE AND FUNCTIONAL EXPRESSION OF A CDNA-ENCODING THE THIAZIDE-SENSITIVE, ELECTRONEUTRAL SODIUM-CHLORIDE COTRANSPORTER [J].
GAMBA, G ;
SALTZBERG, SN ;
LOMBARDI, M ;
MIYANOSHITA, A ;
LYTTON, J ;
HEDIGER, MA ;
BRENNER, BM ;
HEBERT, SC .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1993, 90 (07) :2749-2753
[9]  
GAMBA G, 1994, J BIOL CHEM, V269, P17713
[10]   Molecular cloning and functional expression of the K-Cl cotransporter from rabbit, rat, and human - A new member of the cation-chloride cotransporter family [J].
Gillen, CM ;
Brill, S ;
Payne, JA ;
Forbush, B .
JOURNAL OF BIOLOGICAL CHEMISTRY, 1996, 271 (27) :16237-16244