Dimeric architecture of the human bumetanide-sensitive Na-K-Cl Co-transporter.

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.

Mutations in the human Na-K-2Cl cotransporter (NKCC2) identified in Bartter syndrome type I consistently result in nonfunctional transporters.

Bartter syndrome (BS) is a heterogeneous renal tubular disorder affecting Na-K-Cl reabsorption in the thick ascending limb of Henle's loop. BS type I patients typically present with profound hypokalemia and metabolic alkalosis. The main goal of the present study was to elucidate the functional implications of six homozygous mutations (G193R, A267S, G319R, A508T, del526N, and Y998X) in the bumetanide-sensitive Na-K-2Cl cotransporter (hNKCC2) identified in patients diagnosed with BS type I. To this end, capped RNA (cRNA) of FLAG-tagged hNKCC2 and the corresponding mutants was injected in Xenopus laevis oocytes and transporter activity was measured after 72 h by means of a bumetanide-sensitive (22)Na(+) uptake assay at 30 degrees C. Injection of 25 ng of hNKCC2 cRNA resulted in bumetanide-sensitive (22)Na(+) uptake of 2.5 +/- 0.5 nmol/oocyte per 30 min. Injection of 25 ng of mutant cRNA yielded no significant bumetanide-sensitive (22)Na(+) uptake. Expression of wild-type and mutant transporters was confirmed by immunoblotting, showing significantly less mutant protein compared with wild-type at the same cRNA injection levels. However, when the wild-type cRNA injection level was reduced to obtain a protein expression level equal to that of the mutants, the wild-type still exhibited a significant bumetanide-sensitive (22)Na(+) uptake. Immunocytochemical analysis showed immunopositive staining of hNKCC2 at the plasma membrane for wild-type and all studied mutants. In conclusion, mutations in hNKCC2 identified in type I BS patients, when expressed in Xenopus oocytes, result in a low expression of normally routed but functionally impaired transporters. These results are in line with the hypothesis that the mutations in hNKCC2 are the underlying cause of the clinical abnormalities seen in patients with type I BS.

Functional implications of mutations in the human renal outer medullary potassium channel (ROMK2) identified in Bartter syndrome.

Bartter syndrome is an autosomal recessive heterogeneous renal tubular disorder affecting NaCl reabsorption in the thick ascending limb of Henle's loop (TAL). The aim of this study was to elucidate the functional implications of mutations in the predominant human ROMK isoform in TAL, hROMK2, involved in Bartter syndrome type II. cRNA of flag-tagged hROMK2 and eight mutants identified in seven non-related patients was expressed in Xenopus laevis oocytes. hROMK2 activity was measured by two-electrode voltage-clamp analysis and defined as the Ba2+ -sensitive current at a holding potential of -75 mV. The subcellular localization of hROMK2 in oocytes was studied by immunocytochemistry. Injection of 25 pg hROMK2 cRNA resulted in an inwardly rectifying Ba2+ -sensitive current of 522+/-43 nA ( n=22). The mutants could be divided into three distinct groups. First, at 25 pg injection mutants W80C, V103E and T313/350X exhibited no significant currents and could only be detected intracellularly. Upon 8 ng injection, plasma membrane presence was observed as well as currents up to 60% of wild-type current. Second, mutants V53E and V296G exhibited no Ba2+ -sensitive current, but were present in the plasma membrane at 0.1 ng and 8 ng injection levels. Third, mutants P91L and A179T were detectable on the plasma membrane (0.1 ng) and yielded currents of 98% and 80% of wild-type, respectively, at 25 pg injection. S294C yielded currents that were 45% of wild-type and were detected both on and just below the plasma membrane at 0.1 ng injection. This study has unraveled three distinct mechanisms by which mutations in hROMK2 could impair channel function in Bartter syndrome. Future experiments on kidney epithelial cell lines will have to confirm this classification, after which specific pharmacological treatments could be considered for each group of mutations.