In stable
CCT007093 with wild-type AQP5, AQP5 is clearly expressed on the plasma membrane (Fig. 1A and D). Similar results were obtained in NIH3T3 cells stably transfected with wild-type AQP5 (without FLAG) and stained with C-terminal AQP5 antibody, indicating that fusion of FLAG does not alter AQP5 sub-cellular localization and trafficking to the apical membrane (results not shown). Furthermore, AQP5 is predominantly localized to a 2–2.5 μm thick band at apical pole, consistent with expression in the apical membrane and sub-apical vesicles. We confirmed that AQP5 was polarized to the apical membrane region by double staining experiments in fixed cells. By using a polyclonal antibody against ZO-1, a protein localized to the plasma face of tight junctions, AQP5 was further confirmed to be expressed in the same horizontal plane between tight junctions (Fig. 1A). In stable cells with S156, surprisingly, we observed robust membrane expression of AQP5 (Fig. 1B and E). In ZO-1 co-staining experiment, S156A mutant was localized in the same plane between tight junctions (Fig. 1B), which we subsequently confirmed lack of phosphorylation in this mutant (Fig. 4A). This means that unlike AQP2, even though PKA dependent phosphorylation is blocked, membrane trafficking of AQP5 is not affected. In stable
cells with N185D, AQP5 expression was distributed throughout the whole cytoplasm, not to the apical membrane (Fig. 1C and F). Co-stained ZO-1 was localized above N185D fluorescence, indicating N185D mutant was not translocated to the apical membrane and distributed diffusely throughout cytoplasm (Fig. 1C). Overall, these findings suggest that like AQP1, proper channel formation may be critical for membrane expression of AQP5 and the NPA motif may play a role in protein folding and/or oligomerization that affects trafficking in general.
