The NHE3 Juxtamembrane Cytoplasmic Domain Directly Binds Ezrin: Dual Role In NHE3 Trafficking and Movement In the Brush Border

Epithelial brush border (BB) Na/H antiporter3 (NHE3) associates with the actin cytoskeleton by binding to PDZ domain containing proteins NHERF1‐3. We now show that NHE3 directly binds ezrin (ERM) at a site in its C‐terminus between aa 475–589 separate from the PDZ interacting domain. This is an area predicted to be a‐helical, with a positive aa cluster on one side (K516, R520, and R527). Point mutations of these positively charged aa reduced (NHE3 double mutant (R520F, R527F)) or totally abolished (NHE3 triple mutant (K516Q, R520F, K 527F)) ERM binding. Functional consequences of these NHE3 point mutants included (a) A marked decrease in surface amount and greater decrease in NHE3 activity. (b) Decreased surface expression due to decreased rates of exocytosis and plasma membrane delivery of newly synthesized NHE3, with normal total expression levels and endocytosis rates. (c) A longer plasma membrane half‐life of mutant NHE3 with normal total half‐life. (d) Decreased BB mobile fraction of NHE3 double mutant. These results show that NHE3 binds ERM directly as well as indirectly and direct binding is necessary for trafficking and BB mobility.