Open AccessDimeric Architecture of the Human Bumetanide-Sensitive Na-K-Cl Co-transporter
Author(s) -
Patrick G. Starremans,
Ferry F.J. Kersten,
L.P.W.J. van den Heuvel,
Nine V A M Knoers,
René J. M. Bindels
Publication year - 2003
Publication title -
journal of the american society of nephrology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 4.451
H-Index - 279
eISSN - 1533-3450
pISSN - 1046-6673
DOI - 10.1097/01.asn.0000097370.29737.5b
Subject(s) - xenopus , concatemer , bumetanide , protein subunit , chemistry , wild type , reabsorption , oocyte , transporter , microbiology and biotechnology , biology , biophysics , biochemistry , ion transporter , endocrinology , mutant , membrane , kidney , gene , embryo , genome
. The primary mediator of NaCl reabsorption in the renal distal tubule is the human bumetanide-sensitive Na + -K + -2Cl − co-transporter (hNKCC2), located at the apical membrane of the thick ascending limb of Henle’s loop. The physiologic importance of this transporter is emphasized by the tubular disorder Bartter syndrome type I, which arises from the functional impairment of hNKCC2 as a result of mutations in the SLC12A1 gene. The aim of the present study was to investigate the oligomeric state of hNKCC2 to understand further its operational mechanism. To this end, hNKCC2 was heterologously expressed in Xenopus laevis oocytes. Chemical cross-linking with dimethyl-3,3-dithio-bis-propionamidate indicated that hNKCC2 subunits can reversibly form high molecular weight complexes. Co-immunoprecipitation of tagged hNKCC2 subunits further substantiated a physical interaction between individual hNKCC2 subunits. The size of the hNKCC2 multimers was determined by sucrose gradient centrifugation, and a preference for dimeric complexes (approximately 320 kD) was demonstrated. Finally, concatemeric constructs consisting of two wild-type subunits or a wild-type and a functionally impaired hNKCC2 subunit (G319R) were expressed in oocytes. Subsequently, the concatemers were functionally characterized, resulting in a significant bumetanide-sensitive 22 Na + uptake of 2.5 ± 0.2 nmol/oocyte per 30 min for the wild-type–wild-type concatemer, which was reduced to 1.3 ± 0.1 nmol/oocyte per 30 min for the wild-type–G319R concatemer. In conclusion, this study suggests that hNKCC2 forms at least functional dimers when expressed in Xenopus laevis oocytes of which the individual subunits transport Na + independently.