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Articles in PresS, published online ahead of print May 22, 2002
Am J Physiol Cell Physiol, 10.1152/ajpcell.00613.2001
Submitted on December 26, 2001
Accepted on May 13, 2002
1 GI, Physiology, Cell Biology, Johns Hopkins University School of Medicine, Baltimore, MD, USA
2 Physiology, University of Montreal, Montreal, Quebec, Canada
3 Biology, Curie Institute, Paris, France, France
* To whom correspondence should be addressed. E-mail: mdonowit{at}jhmi.edu.
Cell biologic approaches were used to examine the location and function of the brush border (BB) Na+/H+ exchanger NHE3 in the OK (opossum kidney) polarized renal proximal tubule cell line. Two cell lines were studied: wild-type OK-P cells and OK cells first selected to have a low expression of endogenous NHE3 and then transfected with rat NHE3 epitope tagged with the VSVG epitope (NHE3V). This cell line is called OK-E3V. Both OK-P and OK-E3V cells expressed a similar amount of total NHE3. Based on cell surface biotinylation studies, both cell lines had 10-15% of total NHE3 on the BB. Intracellular NHE3V largely co-localized with Rab11, and to a lesser extent with EEA1. The BB location of NHE3V was examined by confocal microscopy relative to the lectins WGA and PHA-E as well as the B subunit of cholera toxin (CTB). The cells were pyramidal and NHE3 was located in microvilli in the center of the apical surface. In contrast, PHA-E, WGA and CTB subunit were diffusely distributed on the BB, except WGA did not stain the microvilli in the peripheral part of the BB or cell junctions. Detergent extraction of OK cells showed that total NHE3V was largely soluble in Triton X-100, while virtually all surface NHE3V was insoluble. Sucrose density gradient centrifugation demonstrated that total NHE3V migrated at the same size as ~400 and ~900 kDa standards, while surface NHE3V was enriched in the ~900 kDa form. Whether the large sized NHE3 represents its presence in complexes and/or in lipid rafts is not known. Under basal conditions, both endogenous NHE3 and NHE3V cycled between the cell surface and the recycling pathway through a PI 3-kinase dependent mechanism, with the PI 3-kinase inhibitor, wortmannin, decreasing BB NHE3 activity and percent of surface NHE3 by ~50%. Measurements of surface and intracellular pH were obtained using FITC-WGA. Internalization of FITC-WGA occurred largely into the juxtanuclear compartment that contained Rab11 and NHE3V. pH values on the apical surface and in endosomes in the presence of the NHE3 blocker, S3226, were elevated, showing that NHE3 functioned to acidify both compartments. In conclusion, NHE3V exists in distinct domains both in the center of the apical surface and in a juxtanuclear compartment. In the BB fraction it is largely in the detergent insoluble fraction in LR and/or in large heterogenous complexes ranging from ~400 to ~900 kDa. The function of NHE3 in both domains beyond Na+ absorption remains to be determined, as does the mechanism by which NHE3 is maintained in the central apical surface location.
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