Analysis of Na+,K+-ATPase motion and incorporation into the plasma membrane in response to G protein-coupled receptor signals in living cells

Abstract

Dopamine (DA) increases Na(+),K(+)-ATPase activity in lung alveolar epithelial cells. This effect is associated with an increase in Na(+),K(+)-ATPase molecules within the plasma membrane (). Analysis of Na(+),K(+)-ATPase motion was performed in real-time in alveolar cells stably expressing Na(+),K(+)-ATPase molecules carrying a fluorescent tag (green fluorescent protein) in the alpha-subunit. The data demonstrate a distinct (random walk) pattern of basal movement of Na(+),K(+)-ATPase-containing vesicles in nontreated cells. DA increased the directional movement (by 3.5 fold) of the vesicles and an increase in their velocity (by 25%) that consequently promoted the incorporation of vesicles into the plasma membrane. The movement of Na(+),K(+)-ATPase-containing vesicles and incorporation into the plasma membrane were microtubule dependent, and disruption of this network perturbed vesicle motion toward the plasma membrane and prevented the increase in the Na(+),K(+)-ATPase activity induced by DA. Thus, recruitment of new Na(+),K(+)-ATPase molecules into the plasma membrane appears to be a major mechanism by which dopamine increases total cell Na(+),K(+)-ATPase activity.

Figure 1

Incorporation of GFP-NKα₁ into the plasma membrane. (A) Intrinsic GFP-NKα₁ fluorescence in A549 cells grown on coverslips. (B) Na⁺,K⁺-ATPase activity in the presence (DA) or absence (V) of 1 μM DA (5 min at 23°C) was determined in A549 cells expressing either the GFP-NKα₁ (green bars) or the nontagged (black bars) isoform. Each bar represents the mean + SE of four experiments performed in triplicate. (C) GFP-NKα₁ abundance in the basolateral plasma membrane prepared from A549 cells previously treated with (DA) or without (V) 1 μM DA (5 min at 23°C). Na⁺,K⁺-ATPase was detected by Western blot (Laemmli, 1970) with a GFP antibody or (in the same membrane after stripins) with an α-subunit antibody. Equal amount of protein (Bradford, 1976) was analyzed in each lane. (D) Top panel: Low magnification image of the live A549 cell stably expressing GFP-NKα₁. Lower panels: Enlarged time-lapse sequence of region boxed in top panel. First image was acquired 27 s before addition of DA (indicated by the filled arrowhead). Progression of GFP-NKα₁ to the plasma membrane is denoted by the arrowheads. Numbers in the right upper corners indicate time in seconds; scale bar, 5 μm.

Figure 2

Motion of GFP-NKα₁. (A) Projection image was generated by merging successive frames of a stack, collected at 3-s intervals. If the object displays motion its position shifts accordingly in the next frame and on the merge image, it creates a trajectory of the motion (linear if it moves directionally or nonlinear if random-walk displacement occurs). Each projection contains 50 frames before and after addition of DA. Arrows show the starting point. Scale bar, 5 μm. (B) Vesicles were tracked during 100 s before and after addition of DA. Each of the paired tracks (before and after) represents the motion of the same vesicle. Circles on the right side show the average of displacement corresponding to several vesicles. (C) Life histories of the vesicle displacement over time before and after addition of DA. Each line represents a different vesicle. (D) Histograms of velocity and displacement of GFP-NKα₁ (average) before (▪) and after (□) addition of DA. (E) Life histories of the LTG vesicles displacement over time before and after addition of DA. Each line represents a different vesicle. (F) Histograms of velocity and displacement of LTG vesicles (average) before (▪) and after (□) addition of DA.

Figure 3

Effect of MT depolymerization on GFP-NKα₁ motion. (A) Immunostaining of the actin network and MT after nocodazole (NOC) treatment (1 μg/ml for 1 h at 37°C). Red color represents the actin cytoskeleton, green, MT; and blue, GFP-NKα₁ in the merged image. Scale bar, 10 μm. (B) Projection images generated as described in Figure 2B. Scale bar, 5 μm. (C) Life histories of the vesicle displacement in cell treated with NOC before and after DA stimulation. (D) Histograms of velocity and displacement of GFP-NKα₁ (average) in NOC-treated cells before (▪) and after (□) addition of DA. The histograms were created as described in Figure 2E. (E) Changes in Na⁺, K⁺-ATPase activity was examined in A549 cell previously treated with NOC (as in A). Incubation with DA 1 μM or vehicle (Hanks' medium) was performed during 5 min at 23°C. Enzyme activity was expressed as nmol Rb/mg prot/min. Data represents the mean + SE of five experiments in triplicate determinations.

Figure 4

Effect of MT stabilization on GFP-NKα₁ motion. (A) Immunostaining of actin and MT after taxol (TX) treatment (5 μg/ml for 2 h at 37°C). Red color represents actin cytoskeleton, green, MT; blue, GFP-NKα₁ on the merged image. (B) Projection images generated as described in Figure 2B. Scale bar, 5 μm. (C) Life histories of GFP-NKα₁ displacement in cells treated by TX before and after DA stimulation. (D) Histograms of velocity and displacement of GFP-NKα₁ (average) in TX-treated cells before (▪) and after (□) addition of DA. The histograms were created as described in Figure 2E. (E) Changes in Na⁺, K⁺-ATPase activity was examined in A549 cell previously treated with TX (as in A). Incubation with DA 1 μM or vehicle (Hanks' medium) was performed during 5 min at 23°C. Enzyme activity was expressed as nmol Rb/mg prot/min. Data represents the mean ± SE of five experiments in triplicate determinations.

Figure 5

Effect of the actin cytoskeleton on GFP-NKα₁ motion. (A) Immunostaining of actin and MT after latrunculin (LAT) treatment (100 nM for 30 min). Red color represents actin, and cyan MT on the merged image. Scale bar, 10 μm. (B) Histograms of velocity and displacement of GFP-NKα₁ (average) in latrunculin-treated cells before (▪) and after (□) addition of DA (bars). The histograms were created as described in Figure 2D. (C) Changes in Na⁺,K⁺-ATPase activity was examined in A549 cell previously treated with latrunculin (as in A). Incubation with DA 1 μM or vehicle (Hanks' medium) was performed during 5 min at 23°C. Enzyme activity was expressed as nmol Rb/mg prot/min. Data represents the mean ± SE of five experiments in triplicate determination.