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Biochem Biophys Res Commun
2005 May 06;3302:410-4. doi: 10.1016/j.bbrc.2005.02.172.
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Regulation of apical localization of the thiazide-sensitive NaCl cotransporter by WNK4 in polarized epithelial cells.
Yang SS
,
Yamauchi K
,
Rai T
,
Hiyama A
,
Sohara E
,
Suzuki T
,
Itoh T
,
Suda S
,
Sasaki S
,
Uchida S
.
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Missense mutations in the WNK4 gene have been postulated to cause pseudohypoaldosteronism type II (PHAII), an autosomal-dominant disorder characterized by hyperkalemia and hypertension. Previous reports using Xenopus oocytes showed that wild-type WNK4 expression inhibited surface expression of the thiazide-sensitive NaCl cotransporter (NCC), while a disease-causing mutant lost the inhibitory effect on NCC surface expression. To determine if these changes observed in oocytes really occur in polarized epithelial cells, we generated stable MDCK II cell lines expressing NCC alone or NCC plus wild-type WNK4 or a disease-causing (D564A) WNK4. In contrast to the apical localization of NCC without co-expression of WNK4, immunofluorescence microscopy and biotin surface labeling revealed that this apical localization was equally decreased by both the wild-type and the mutant WNK4 expression. Apical localizations of two PHAII-unrelated apical transporters, sodium-independent amino acid transporter, BAT1 and bile salt export pump, Bsep, were also found to be decreased by both wild-type and mutant WNK4 expression. These results indicate that the regulation of NCC was not related to the disease-causing mutation and not restricted to the PHAII-related specific transporters. The regulation of intracellular localization of NCC by WNK4 might not be involved in the pathogenesis of PHAII.
Fig. 1.
Generation of MDCK II cell lines that express NCC alone or co-express NCC and wild-type or mutant (D564A) WNK4. (A) HA-tagged NCC proteins and (B) Flag-tagged WNK4 proteins were fractioned by SDSâPAGE and detected by using an anti-HA mAb (9Y10) and mouse M2 anti-Flag mAb, respectively. Expression of NCC and WNK4 protein was induced by removal of doxycycline (Dox) from the medium for 4 days. 1, host cell; 2 and 3, cell line expressing NCC only; 4 and 5, cell line co-expressing NCC and wild-type WNK4; and 6 and 7, cell line co-expressing NCC and mutant WNK4.
Fig. 2.
Cellular localization of NCC in the established cell lines. Localization of stably expressed NCC in MDCK II cells expressing NCC only: (1) or co-expressing NCC and wild-type WNK4 (2) or mutant WNK4 (3). XY (A) and XZ (B) images were obtained by confocal laser scanning microscopy. Immunocytochemistry was performed with rat anti-HA mAb (9Y10) and mouse anti-β catenin mAb as primary antibodies, and anti-rat IgG Alexa 546 (red) and anti-mouse IgG Alexa 488 (green) as the secondary antibodies. The arrowhead indicates the apical cell surface.
Fig. 3.
Cell surface biotinylation assay. Apical cell surface expression of NCC in MDCKII cells expressing NCC only (1) or co-expressing NCC and wild-type WNK4 (2) or mutant WNK4 (3). Stable cells expressing NCC and WNK4 were subjected to apical cell surface biotinylation assay. Biotinylated proteins were isolated with streptavidin-agarose beads and immunoblotted for NCC by anti-HA mAb (9Y10). Apical biotinylated proteins (upper panel) and the total cell lysates (middle panel) were fractioned by SDSâPAGE and immunoblotted.
Fig. 4.
Localization of GFP-BAT1 and Bsep transiently expressed in MDCK II cells. Immunofluorescence of green fluorescent protein (GFP)-tagged BAT1 (upper panels) and Bsep (lower panels) in host cells (1) and MDCK II cells expressing wild-type WNK4 (2) or mutant WNK4 (3). BAT1 is shown in green and β-catenin is shown in red (upper panels). Bsep is shown in red and β-catenin is shown in green (lower panels). Arrowheads indicate the apical cell surface.
