FIGURE 4.

Function of NHE3-Ser⁵¹⁵, -Ser⁵²², and -Ser⁵²⁶ in NHE3 activity in basal conditions and with Akt_i in PS120 and OK cells. A, PS120 cell lines stably expressing wild type NHE3 or mutated NHE3 (S515A/D, S522A/D, and S526A/D) were generated. NHE3 activity with or without Akt_i was measured with BCECF/fluorometry. Expression of each cell line is shown in the inset with actin for normalization. NHE3-S515A and -S515D single mutants had lower activity than wild type NHE3 and did not respond to Akt_i. Both NHE3-S522A and -S522D had lower basal activity but maintained Akt_i inhibition. NHE3-S526D had higher activity than NHE3-S526A. The Akt_i inhibition of NHE3 activity was not present in NHE3-S526A cells but occurred similarly to wild type in NHE3-S526D cells. B, studies as in A (including the level of expression shown in inset) were performed in OK cells stably transfected with wild type and mutated NHE3. The results in OK cells gave the same pattern of results described in PS120 cells in A. The means ± S.E. are from at least six separate studies. p values represent Akt_i effect compared with untreated control and are shown only when p ≤ 0.05. IB, immunoblot.