Fig. 7.

The AMPK phosphorylation-deficient S384A V-ATPase A subunit mutant remains at the apical membrane of IC in response to an AMPK activator. Clone C cells were transiently transfected with either the WT-A or S384A-A subunit. A: 1 day after transfection with either the WT-A (top) or S384A-A mutant subunit (bottom), clone C cells were plated onto Transwell filters. After 4 days, the filters were incubated for 4 h in media (left) or in media with 2 mM AICAR (right). Filters were then incubated with concanavalin A coupled to CY3 (red), fixed, and immunofluorescently labeled using anti-FLAG antibody (green) and TO-PRO-3 nuclear stain (blue). Scale bar = 10 μM. B: quantification of V-ATPase-associated MPI ratio of apical region of interest (ROI-1; here the A subunit colocalizes with concanavalin A) and cytoplasmic ROI-2 (A subunit alone). This ROI-1/ROI-2 ratio under the different conditions reveals a significant AICAR-mediated inhibition of apical V-ATPase accumulation in cells expressing the WT A subunit compared with cells expressing the S384A mutant. Values are means ± SE. *P < 0.05 vs. WT-A Control. #P < 0.05 vs. WT-A AICAR; n = 20–45 cells analyzed for both conditions. C: representative immunoblots of pThr-172-AMPKα and β-actin in clone C cell lysate filters treated with media alone (left) or with media+AICAR (right). D. quantification of the ratio of pThr-172-AMPK-α to β-actin Western blot signals shown in C normalized to Control as a measure of relative AMPK activity. *P < 0.05; n = 3.