FIGURE 3.

Acute elevation of Ca²⁺ dynamically increases the lateral mobility of NHE3 determined by FRAP in the apical domain of OK cells. A, 4-Br-A23187 inhibition of NHE3 activity does not occur with exposure to K⁺-deficient medium. Exposure to K⁺-deficient medium (142 mm NaCl, 1 mm CaCl₂, 1 mm MgCl₂, 20 mm HEPES, pH 7.4, pH adjusted with Tris) was for 1 h before transport measurements. K⁺-deficient medium caused a slight but not significant decrease in basal NHE3 activity. Results shown are mean ± S.E. from three experiments. p values are paired t tests. B, dynasore pretreatment (80 μm, 30 min) slightly but not significantly lowered basal NHE3 activity and prevented A23187 inhibition of NHE3. Results are mean ± S.E. from three experiments. p values are paired t test. C, FRAP studies of NHE3 were performed on OK/NHERF2 cells transiently transfected with NHE3-EGFP with/without (control) 4-Br-A23187 (0.5 μm) starting 30 min after A23187 exposure. Relative fluorescence intensity after photo-bleaching was measured for 300 s. K⁺-deficient medium, as above, was used to reduce endocytosis induced by elevated Ca²⁺ (30). Inset images show NHE3-EGFP pre-bleaching (C1), immediately post-bleaching (C2), and after recovery (300 s) (C3) in studies performed 30 min after A23187 exposure. D, elevated Ca²⁺ transiently increased NHE3-EGFP mobility in OK/NHERF2/NHE3-EGFP cells. FRAP was performed before and at various times after 4-Br-A23187 addition to measure BB lateral mobility of NHE3-EGFP in OK cells. This was done with K⁺-deficient medium pretreatment for 1 h and throughout the experiment. Minutes after A23187 addition when FRAP studies were performed is shown on the x axis. Results of a typical experiment are shown with mean ± S.E. shown for ROIs from this single experiment to show the variability with at least three ROIs from >1 cell analyzed. E, Ca²⁺ elevation via A23187 transiently stimulates NHE3 mobile fraction at 30 min, which then returns to base line at 60 min under K⁺-deficient conditions as well as with dynasore (80 μm, 30 min) treatment. Basal NHE3 mobile fraction with normal medium or K⁺-deficient medium or dynasore without 4-Br-A23187 did not change significantly over the time of the experiments. Similar results were found with both K⁺-deficient media exposure and dynasore. The mean ± S.E. were calculated from three independent experiments. p values are in comparison to zero time (paired t test). F, A23187 did not alter the NHE3 mobile fraction 30 min after A23187 addition in the absence of NHERF2. Study was done with K⁺-deficient medium as in D. Results are mean ± S.E. of 30-min time point from three experiments. NS, not significant.