The epithelial Na(+)/H(+) exchanger, NHE3, is internalized through a clathrin-mediated pathway - PubMed (original) (raw)

. 1999 Dec 31;274(53):37551-8.

doi: 10.1074/jbc.274.53.37551.

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The epithelial Na(+)/H(+) exchanger, NHE3, is internalized through a clathrin-mediated pathway

C W Chow et al. J Biol Chem. 1999.

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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 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.

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