The epithelial Na(+)/H(+) exchanger, NHE3, is internalized through a clathrin-mediated pathway.
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 clathrin-mediated 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 epsilon-COP subunit. At the permissive temperature, NHE3 distributed normally, whereas at the restrictive temperature, which induces rapid degradation of epsilon-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 epsilon-COP-dependent step.
Chow
C W
CW
Division of Respiratory Medicine, Department of Medicine, University of Toronto, Toronto, Ontario M5S 1A8, Canada.
Khurana
S
S
Woodside
M
M
Grinstein
S
S
Orlowski
J
J
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002966:Clathrin / Q000378:metabolism*; D006224:Cricetinae; D004705:Endocytosis; D004847:Epithelial Cells / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D009994:Osmolar Concentration; D011188:Potassium / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D017923:Sodium-Hydrogen Exchangers / Q000378:metabolism*
D002966:Clathrin; D011994:Recombinant Proteins; D017923:Sodium-Hydrogen Exchangers; D011188:Potassium