FIGURE 4.

NHE3 and NHERF3 dissociate after elevation of intracellular calcium in PS120 cells. A, PS120/NHE3V/NHERF3 cells were seeded onto glass coverslips and grown to 70–80% confluence. Cells were serum starved for 4 h and treated with either vehicle or 4-bromo-A23187 (0.5 μm) for 15 min at 37 °C. Cells were then fixed with 3% paraformaldehyde, permeabilized, and stained with antibodies to NHE3 and NHERF3 and localization determined by confocal microscopy. In PS120/NHE3V/NHERF3 cells, NHE3 and NHERF3 colocalize in distinct areas within the plasma membrane as well as within intracellular vesicles. B, after elevation of intracellular calcium, NHE3 and NHERF3 no longer colocalize either at the plasma membrane or in intracellular compartments (×100 oil objective lens; 0.5-μm confocal sections). Insets magnify (×3 zoom) areas along the plasma membrane. Results were obtained from six to eight individual experiments.