WNK3 interacts with NCC

Abstract

Dear Editor, NCC (Na-Cl cotransporter) is a cotransporter mainly distributed in the distal tubule of the kidney, functioning to reabsorb sodium and chloride ions from the tubular fluid into the cells of the renal distal convoluted tubule. It is a transmembrane protein belonging to the SLC12 cotransporter family of electro-neutral cation-coupled chloride cotransporters, which is closely related to hypertension (Gamba, 2005). A loss of NCC function can cause Gitelman syndrome, a disease characterized by low blood pressure, hypocalciuria, hypokalemic metabolic alkalosis, and hypomagnesemia, etc. WNK3 (With No Lysine K 3) is highly expressed in the whole length of the renal tubules (Glover et al., 2009). It was previously found to enhance the activity of NCC in Xenopus oocyte (Rinehart et al., 2005). Exploring the regulatory effect of WNK3 on NCC may be helpful to understand the occurrence of abnormal renal sodium transporters and provide new targets for the treatment of hypertension. However, whether WNK3 interacts directly with NCC in mammalian cells still remains unclear. In this article, we report the results of our co-immunoprecipitation experiments and confocal observations, confirming that WNK3 does interact with NCC and that the kinase domain of WNK3 plays an important role in the regulation of NCC.To explore whether there is reaction between WNK3 and NCC protein, Cos-7 cells were transfected by GFP-NCC with or without HA-WNK3 and co-immunoprecipitation experiments were performed 48 hours after transfection. As shown in Figure 1, anti-HA antibody with the presence of WNK3 immunoprecipitated NCC whereas serum with the absence of WNK3 did not, suggesting that the WNK3 protein interacts with NCC directly.In order to investigate the effect of WNK3 on NCC, HA-NCC was transfected into Cos-7 cells while increasing doses of HA-WNK3. Western-blot was carried out with anti-HA or anti-GAPDH antibodies 48 h later. The NCC protein expression level gradually increased with the increase of WNK3 dosage ( Figure S1 in Supporting Information). The expression of NCC and WNK3 was checked under a confocal fluorescent microscope ( Figure S2 in Supporting Information). The results showed that WNK3 increased NCC expression both on the cell membrane and in the cytoplasm. It has been confirmed that the enzyme region, the amino terminal, and/or the carboxyl terminal are the key elements of kinase activity. However, whether the enzyme region of WNK3 is necessary in regulating NCC is still unclear. We observed the changes of NCC expression after various WNK3 fragments were transferred into the cells. As shown in Figure S3 in Supporting Information, only the WNK3 fragments containing the enzyme region were able to increase the expression of NCC.WNK kinases belong to a subfamily of serine/threonine kinases. It was reported that the activity of NCC could be down-regulated by WNK4 (Moniz et al., 2010). Our study showed that in the mammalian cells WNK3 was also a chloride sensitive kinase to NCC expression, with its effect being the opposit...

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