PMC

Effect of monensin (Mon) and dopamine (DA) on Na-K-ATPase-mediated Rb⁺ uptake. Opposum kidney (OK) cells expressing the wild-type (WT, left) or S18A mutant form of Na-K-ATPase-α1 (right) were treated with 5 μM monensin for 30 min and 1 μM dopamine for 5 min before Rb⁺ uptake assay. *P < 0.05.

Images of cells expressing green fluorescent protein (GFP)-tagged α1 observed under epifluorescence and total internal reflection fluorescence (TIRF) illumination. A: brightfield image of a typical OK cell taken using Hoffman modulation contrast optics. B and C: fluorescence images of the same cell acquired using epi- and evanescent wave illumination, respectively. Bar = 5 μm.

Colocalization of Na-K-ATPase-α1 and type 1 dopamine receptor (D1R). Images of OK cells expressing Na-K-ATPase wild-type α1 treated with vehicle (top), 5 μM monensin for 30 min (middle), and 5 μM monensin for 30 min, and then with 1 μM dopamine for 5 min (bottom) are shown. Fluorescence images were acquired through the Cy3 channel (Na-K-ATPase-α1) and the Alexa Fluor 488 channel (D1R). Merged images (right) were obtained using SimplePCI software. Cells were treated and images acquired as described in materials and methods. Arrows point to the sites of colocalization.

Effect of monensin and dopamine on plasma membrane Na-K-ATPase-α1 abundance. OK cells expressing the wild-type or S18A mutant form of Na-K-ATPase-α1 were treated with 5 μM monensin for 30 min and 1 μM dopamine for 5 min. The abundance of Na-K-ATPase molecules at the plasma membrane was determined by biotinylation, as described in materials and methods. After biotinylation, the cells were dissolved and, from samples containing the same amount of protein, Na-K-ATPase molecules were precipitated using a specific antibody. The amount of biotin present in the immunoprecipitated Na-K-ATPase molecules was determined by Western blot analysis. Top: representative Western blots. Bottom: quantitation of Western blots. *P < 0.05 with respect to cells treated with monensin alone.

Increased TIRF level of GFP-tagged Na-K-ATPase-α1 in response to increased intracellular sodium concentration. A: control fluorescence image and corresponding pixel-by-pixel fluorescence determination of an OK cell obtained by evanescent wave illumination. Bar = 5 μm. B: TIRF images obtained at different sodium concentrations after the cell membrane was permeabilized with 10 μM gramicidin D. C: percentage of signal changes (means ± SD) observed in relation to control fluorescence levels from 18 different cells. Under basal conditions, these cells have an average of 9 mM intracellular sodium (). Numbers on the abscissa indicate the increase of intracellular sodium with respect to the control basal intracellular sodium concentration level.

Fluorescence light changes under evanescent wave illumination evoked in OK cells by monensin and dopamine. TIRF determinations were performed in cells expressing either Na-K-ATPase-α1 (A and B) or the S18A mutant of this protein (C and D), both fused to GFP. Determinations illustrated in the histograms were performed every 2 min (B and D), and arrows indicate the times images illustrated in A and C were captured. Times of application of monensin and dopamine are indicated. Fluorescence increases were coded in pseudocolors and overlapped on a brightfield image of the corresponding OK cell acquired using Hoffman modulation contrast. Blue, green, yellow, and red represent signal increases equivalent to 1, 1.5, 2, 2.5, and 3 SD of the pixel variability in the image background, respectively. Bar = 5 μm.