Expression and trafficking of the gamma subunit of Na,K-ATPase in hypertonically challenged IMCD3 cells

Abstract

The gamma subunit (FXYD2) of Na,K-ATPase is an important regulator of the sodium pump. In this investigation we have analysed the trafficking of gamma to the plasma membrane in cultures of inner medullary collecting duct cells (IMCD3) following acute hypertonic challenge and brefeldin A (BFA) treatment. Following hypertonic challenging for 24 hr immunofluorescence labeling revealed initial co-localization of the gamma subunit and 58K Golgi protein in the cytoplasm, but no co-localization of alpha1 and Golgi protein. Exposure of the challenged cells to BFA prevented the subsequent incorporation of gamma into the basolateral plasma membrane. The gamma subunit instead remained in cytoplasmic vesicles while cell proliferation and cell viability decreased simultaneously. Following removal of BFA from the hypertonic medium the IMCD3 cells recovered with distinct expression of gamma in the basolateral membrane. The alpha1 subunit was only marginally influenced by BFA. The results demonstrate that the gamma subunit trafficks to the plasma membrane via the Golgi apparatus, despite the absence of a signal sequence. The results also suggest that the gamma and alpha subunits do not traffic together to the plasma membrane, and that the gamma and alpha subunit have different turnover rates during these experimental conditions.

Keywords: FXYD2; Golgi apparatus; brefeldin A; confocal microscopy; hypertonicity.

Fig. 1

A: IMCD3 cell proliferation is retarded in hypertonic media. Cells were seeded in two 24-well plates in 300 mOsm/kgH₂O medium at a concentration of 10,000 cells/well. After subconfluences, 3 wells in each plate were counted at the starting point (0 hr) and daily up to 168 hr. Cells on one plate continued to grow in 300 mOsm/kgH₂O medium, while cells on the other plate were challenged with hypertonic 550 mOsm/kgH₂O medium, which significantly retarded cell proliferation. B: IMCD3 cell proliferation is retarded with BFA. IMCD3 cells were seeded on two 24-well plates at a concentration of 200,000 cells/well. When the cells were subconfluent, the cell numbers were counted and this time was defined as the starting point (0 hr). The wells were then divided into 4 groups with different media: 300 mOsm/kgH₂O, 550 mOsm/kgH₂O, 550 mOsm/kgH₂O+1 µM BFA, 550 mOsm/kgH₂O+5 µM BFA. Cell numbers were determined after 24 hr and BFA-media were changed to BFA-free media for another 48 hr. All pairwise comparisons between the curves are significantly different (p<0.001) and illustrate that BFA causes distinct decreases in cell proliferation. C: Cell viability decreases after treatment with BFA. IMCD3 cells were seeded on two 24-well plates at a concentration of 10,000 or 25,000 cells/well. When the cells were subconfluent, 3 wells were split, the number of cells counted, and this time determined as the starting point. The IMCD3 cell viability based on the non-radioactive cell viability test decreased following BFA treatment both in 300 and 550 mOsm/kgH₂O media. Graphs in all figures show mean values±SE.

Fig. 2

Localization of Golgi proteins in IMCD3 cells. Immunolocalization of Golgi proteins GM130 (A, C) and Golgi 58k (B) in IMCD3 cells, cultured in isotonic medium, and acutely challenged with 550 mOsm/kgH₂O medium (C). In 300 mOsm/kgH₂O medium both the GM130 protein (green) (A) and 58k protein (red) (B) are strongly expressed in the Golgi apparatus located close to the nucleus. Nuclei are stained with To-Pro-3. Bar=20 µm.

Fig. 3

Simultaneous acute hypertonic challenge and BFA treatment redistributes Na,K-ATPase γ subunit and Golgi proteins in IMCD3 cells. Over-view images (A–C) and high magnification images (F, I and L) of double-immunolocalized 58K Golgi protein and the γ subunit of Na,K-ATPase in the cytoplasm of IMCD3 cells acutely challenged for 24 hr with 550 mOsm/kgH₂O medium (A and F), simultaneously treated with 1 µM brefeldin A (BFA) (B and I), and subsequently again in 550 mOsm/kgH₂O without BFA (C and L). The γ subunit is strongly expressed in the plasma membrane in clusters of challenged cells (green) (A), very little or not at all expressed in BFA treated cells (B) but again intensely expressed in large clusters of cells during a recovery period in hypertonic medium without BFA treatment (C). This is further demonstrated in high magnification single color and overlay images: A stripe of cells with γ label (green) in the plasma membrane as well as in the cytoplasm of the same IMCD cells (arrows, D). 58K Golgi protein labeling (red) is present in all cells. Golgi areas in the γ-positive cells marked with arrows (E). Co-localization (yellow) in the γ-positive cells in a merged image (arrows, F). In the BFA treated cells γ is lacking in the plasma membrane but is present differently in the cytoplasm of some cells (arrows, G). The expression of 58K Golgi protein is diminished and diffuse in the cytoplasm (H) and co-localizes with γ in overlays (arrows, I). The γ subunit is strongly expressed in the plasma membrane of cells recovered from the BFA treatment in the hypertonic medium for 48 hr and expressed also in the cytoplasm (J). These cytoplasmic areas are also labeled for Golgi 58K and show resemblance to the Golgi apparatus (K, red) and are co-localizing with the γ label (L, arrows). Nuclei are stained with To-Pro-3. Bars=40 µm (A), 15 µm (B). Magnification is the same in A–C, and D–L, respectively.

Fig. 4

Simultaneous acute hypertonic challenge and BFA treatment does not redistribute Na,K-ATPase α1 subunit. Hypertonically challenged (A, C and E) and simultaneously BFA-treated (B, D and F) IMCD3 cells with double-immunostaining for the α1 subunit of Na,K-ATPase (green, A and B) and the 58K Golgi protein (red, C and D) show no co-localization in the overlay images (E and F). Immunolabeling for the α1 subunit in the plasma membrane remains strong after BFA treatment (B). The 58K Golgi protein label is visible in all control cells (C) but only in a few experimental cells (arrows, D). Cell nuclei are stained with To-Pro-3. Same magnification in all images. Bar=15 µm.

Fig. 5

Effect of acute hypertonic challenge and consecutive BFA treatment vs. hypertonic challenge without BFA on the location of Na,K-ATPase γ subunit. Immunolocalization of the γ subunit shows strong plasma membrane staining in IMCD3 cells acutely challenged in 550 mOsm/kgH₂O medium for 48 hr (control) (A) and at higher magnification (B). First hypertonic challenging (24 hr) and then 1 µM BFA in 550 mOsm/kgH₂O medium (24 hr) results in absence of γ from the plasma membrane but diffuse or vesicular labeling in the cytoplasm (C and D). In some cells small vesicles are located like pearls in string close to the plasma membrane (arrows, D). Continuous hypertonic challenging for 4 d without BFA does not induce more γ-stained cells than cultures challenged for two days (E cf. A) but indicates strong plasma membrane location (F). The ability of cells to recover after the 1 µM BFA treatment is demonstrated in G and H. Cells received the same BFA treatment as in C and D, but were then incubated in 550 mOsm/kgH₂O medium for 48 hr without BFA (G). The γ subunit is observed in more cells than in the cultures that have been in hypertonic medium only for four days (E). High magnification image (H) demonstrates that γ is present both in the plasma membranes and in the cytoplasm. Cell nuclei are stained blue with To-Pro-3. Magnification is the same in A, C, E and G; bar in A=40 µm. Magnification is the same in B, F and H; bar in B=20 µm. Bar in D=10 µm.

Fig. 6

Different responses of Na,K-ATPase α1 and γ subunits in hypertonic challenged IMCD3 cells with or without BFA treatment. Immunolocalization after acute hypertonic challenge of IMCD3 cells for 48 hr with the monoclonal antibody against the α1 subunit demonstrates uniform location of α1 in the plasma membranes of all cells, while only a portion of cells (γ-negative cells marked with stars in A) shows immunolocalization with polyclonal antibody against the Na,K-ATPase γ subunit (B). Hypertonic challenging for 24 hr and then BFA in a hypertonic medium for 24 hr results in distinct location of α1 (C) in the plasma membrane in most cells and additional weak diffuse location in the cytoplasm, while γ is almost exclusively located diffusely in the cytoplasm of some cells (D). IMCD3 cells in (E) and (F) immunostained for α1 and γ, respectively, were treated as in C and D but subsequently allowed to recover in 550 mOsm/kgH₂O medium without BFA for 48 hr. Similar location of α1 in the plasma membrane as in control (E cf. A), while γ is located both in the plasma membrane but still also diffusely in the cytoplasm (F). Cell nuclei are stained blue with To-Pro-3. Magnification is the same in all images. Bar=20 µm.