FIGURE 4:

CK2 phosphorylation of NHE3-S719 is necessary for Ca²⁺ inhibition of NHE3 but not for inhibition of NHE3 by forskolin (FSK) and hyperosmolarity. (A) FSK (25 µM for 30 min) inhibits NHE3 activity in both NHE3-WT and S719A mutant cells by a similar percentage of basal activity. (B) Carbachol (CCh; 25 µM for 5 min) inhibits NHE3 activity in NHE3-WT cells by ∼40% but had no significant effect in NHE3-S719A cells. (C) NHE3-WT cells treated with calcium ionophore A23187 (1 µM for 10 min) reduced NHE3 activity by ∼30% (p < 0.05), but the effect of A23187 on NHE3-S719A cells was not significant. (D) Treatment of NHE3-WT cells with CCh or DMAT reduced NHE3 activity ∼40%, but there was no additional inhibition of NHE3 when DMAT-treated cells were further treated with CCh. (E) Under hyperosmolar conditions, NHE3 activity was reduced by ∼50% in both NHE3-WT and NHE3-S719A cells (p < 0.05). n, number of experiments. p values are by paired t tests and ANOVA.