The Epithelial Na+/H+ Exchanger, NHE3, Is Internalized through a Clathrin-mediated Pathway

Abstract

Trafficking of the Na ؉ /H؉ exchanger isoform 3 (NHE3) between sub-apical vesicles and apical membrane of epithelial cells is a suggested mechanism of regulation of NHE3 activity. When epitope-tagged NHE3 was stably expressed in NHE-deficient Chinese hamster ovary cells, a sizable fraction was found in recycling endosomes. This system was used to analyze the mechanism of endocytosis of NHE3. Immunofluorescence and radiolabeling experiments showed that inhibition of clathrinmediated endocytosis using hypertonicity, acid treatment, or K ؉ depletion inhibited internalization of NHE3. Moreover, transient transfection of an inhibitory mutant of dynamin (DynS45N) blocked the clathrin-mediated uptake of transferrin, as well as the endocytosis of NHE3. In ileal villus cells, endogenous NHE3 was also found to co-purify with isolated clathrin-coated vesicles, thereby confirming their association in native tissues. The role of COP-I subunits in the intracellular traffic of NHE3 was evaluated using ldlF cells, which bear a temperature-sensitive mutation in the ⑀-COP subunit. At the permissive temperature, NHE3 distributed normally, whereas at the restrictive temperature, which induces rapid degradation of ⑀-COP, NHE3 was still internalized, but its subcellular distribution was altered. These results indicate that endocytosis of NHE3 occurs primarily via clathrin-coated pits and vesicles and that normal intracellular trafficking of NHE3 involves an ⑀-COP-dependent step.

Bimodal Acute Effects of A1 Adenosine Receptor Activation on Na+/H+ Exchanger 3 in Opossum Kidney Cells

“…It was recently shown that endocytosis of NHE3 occurs primarily via clathrin-coated pits (36,37) and that clathrin-mediated endocytosis requires an intact actin cytoskeleton (38). In support of trafficking, we demonstrated that the decrease of NHE3 activity induced by CPA is inhibited by cytochalasin B, which induces cytoskeletal disorganization (31) and reportedly has per se an inhibitory effect on NHE3 (31) (for effect of cytochalasin B, see also the Results section).…”

Acute Regulation of Na/H Exchanger NHE3 by Adenosine A1 Receptors Is Mediated by Calcineurin Homologous Protein

“…CHP1 is involved in targeting/fusion of transcytotic vesicles with the apical membrane (25). Although, NHE3 is endocytosed into clathrin-coated vesicles (40,71), exactly how CHP regulates NHE3 trafficking is unclear.…”

Ca2+-dependent Inhibition of Na+/H+ Exchanger 3 (NHE3) Requires an NHE3-E3KARP-α-Actinin-4 Complex for Oligomerization and Endocytosis

“…Endocytosis has been implicated previously (17,18) in regulation of NHE3 activity by alteration of the amount of NHE3 localized in the plasma membrane. The intracellular localization of NHE3 in the recycling compartment was demonstrated both in nonepithelial cells, PS120 and AP-1 fibroblasts, and in epithelial cells, including Caco-2 and OK epithelial cells (17,18,30,40).…”

“…Another mechanism for acute regulation of NHE3 activity includes membrane trafficking between an intracellular recycling compartment and the plasma membrane (17)(18)(19). NHE3 is active on the plasma membrane, and the alteration of surface NHE3 protein abundance plays a role in regulation of NHE3 activity by extracellular agonists including peptide hormones, growth factors, and neurotransmitters (1).…”