Functional Expression of the Na-K-2Cl Cotransporter NKCC2 in Mammalian Cells Fails to Confirm the Dominant-negative Effect of the AF Splice Variant

Abstract

The renal bumetanide-sensitive Na-K-2Cl cotransporter (NKCC2) is the major salt transport pathway in the apical membrane of the mammalian thick ascending limb. It is differentially spliced and the three major variants (A, B, and F) differ in their localization and transport characteristics. Most knowledge about its regulation comes from experiments in Xenopus oocytes as NKCC2 proved difficult to functionally express in a mammalian system. Here we report the cloning and functional expression of untagged and unmodified versions of the major splice variants from ferret kidney (fNKCC2A, -B, and -F) in human embryonic kidney (HEK) 293 cells. Many NKCC2 antibodies used in this study detected high molecular weight forms of the transfected proteins, probably NKCC2 dimers, but not the monomers. Interestingly, monomers were strongly detected by phosphospecific antibodies directed against phosphopeptides in the regulatory N terminus. Bumetanide-sensitive 86Rb uptake was significantly higher in transfected HEK-293 cells and could be stimulated by incubating cells in a medium containing a low chloride concentration prior the uptake measurements. fNKCC2 was less sensitive to the reduction in chloride concentration than NKCC1. Using HEK-293 cells stably expressing fNKCC2A we also show that co-expression of variant NKCC2AF does not have the dominant-negative effect on NKCC2A activity that was seen in Xenopus oocytes, nor is it trafficked to the cell surface. In addition, fNKCC2AF is neither complex glycosylated nor phosphorylated in its N terminus regulatory region like other variants.

Regulation of the NKCC2 ion cotransporter by SPAK-OSR1-dependent and -independent pathways

“…7, top panel). These observations are consistent with previous reports that also concluded that isoforms 797 Regulation of NKCC2 by SPAK and OSR1 A and B were more active than isoform F when expressed in mammalian cells (Hannemann et al, 2009) or in Xenopus laevis oocytes (Gimenez et al, 2002;Plata et al, 2002). The only differences in the three isoforms of NKCC2 lie in an alternatively spliced exon encoding the proposed second transmembrane domain and a portion of the adjacent first intracellular connecting loop (Castrop and Schnermann, 2008) (Fig.…”

“…7, upper panel). Another recent study also reported that NKCC2 expressed in mammalian cells was not activated upon exposure to hypotonic low-chloride conditions (Hannemann et al, 2009). These differences might suggest that the Xenopus laevis oocyte expression system may be optimal for studying NKCC2 activation.…”

“…In order to compare the activity of different NKCC2 isoforms, we measured bumetanide-sensitive uptake of 86 Rb in HEK-293 cells overexpressing these enzymes at similar levels. Assays were performed in the presence of ouabain to inhibit the activity of the Na + /K + -ATPase, employing a similar approach to that described in a recent study (Hannemann et al, 2009). In cells not transfected with NKCC2, a low level of bumetanide-sensitive 86 Rb uptake was observed, which was increased approximately threefold by hypotonic low-chloride stimulation of cells, presumably owing to activation of the endogenous NKCC1 that is expressed in these .…”

Rare mutations in the human Na-K-Cl cotransporter (NKCC2) associated with lower blood pressure exhibit impaired processing and transport function

“…As a more appropriate test of human NKCC2A function, we sought to characterize the nine mutants in a human cell line. To date, only a few studies have been successful in achieving functional expression of NKCC2 in mammalian cell lines (1,11,29). In our own laboratory, we have found that rabbit NKCC2 is very difficult to express and maintain in mammalian cell culture unless fused to the NKCC1 N terminus (2,13).…”

Interactions with WNK (With No Lysine) Family Members Regulate Oxidative Stress Response 1 and Ion Co-transporter Activity

“…Tonicity was increased or hypertonicity was induced by the addition of 0.2 M sorbitol for 5 min or 0.5 M sorbitol for 30 min; hypotonic conditions were generated by placing cells for 20 min in the medium above diluted 4-fold with water or in medium in which chloride was largely replaced with gluconate: 67.5 mM sodium gluconate, 0.5 mM CaCl 2 , 0.5 mM MgCl 2 , 5 mM glucose, 15 mM HEPES, pH 7.4 (modified from Ref. 41). FRAP was carried out using the 488-nm laser line of a Zeiss LSM780 on an AxioExaminer upright stand with a 20ϫ W/1.0 dipping lens.…”