NHE3 activity is dependent on direct phosphoinositide binding at the N terminus of its intracellular cytosolic region

The small intestinal BB Na⁺/H⁺ antiporter NHE3 accounts for the majority of intestinal sodium and water absorption. It is highly regulated with both postprandial inhibition and stimulation sequentially occurring. Phosphatidylinositide 4,5-bisphosphate (PI(4,5)P₂) and phosphatidylinositide 3,4,5-trisphosphate (PI(3,4,5)P₃) binding is involved with regulation of multiple transporters. We tested the hypothesis that phosphoinositides bind NHE3 under basal conditions and are necessary for its acute regulation. His₆ proteins were made from the NHE3 C-terminal region divided into four parts as follows: F1 (amino acids 475-589), F2 (amino acids 590-667), F3 (amino acids 668-747), and F4 (amino acids 748-832) and purified by a nickel column. Mutations were made in the F1 region of NHE3 and cloned in pet30a and pcDNA3.1 vectors. PI(4,5)P₂ and PI(3,4,5)P ₃ bound only to the NHE3 F1 fusion protein (amino acids 475-589) on liposomal pulldown assays. Mutations were made in the putative lipid binding region of the F1 domain and studied for alterations in lipid binding and Na ⁺/H⁺ exchange as follows: Y501A/R503A/K505A; F509A/R511A/R512A; R511L/R512L; R520/FR527F; and R551L/R552L. Our results indicate the following. 1) The F1 domain of the NHE3 C terminus has phosphoinositide binding regions. 2) Mutations of these regions alter PI(4,5)P₂ and PI(3,4,5)P₃ binding and basal NHE3 activity. 3) The magnitude of serum stimulation of NHE3 correlates with PI(4,5)P ₂ and PI(3,4,5)P₃ binding of NHE3. 4) Wortmannin inhibition of PI3K did not correlate with PI(4,5)P₂ or PI(3,4,5)P₃ binding of NHE3. Two functionally distinct phosphoinositide binding regions (Tyr⁵⁰¹-Arg⁵¹² and Arg⁵²⁰-Arg⁵⁵²) are present in the NHE3 F1 domain; both regions are important for serum stimulation, but they display differences in phosphoinositide binding, and the latter but not the former alters NHE3 surface expression.