Na(+)/H(+) exchanger 3 is in large complexes in the center of the apical surface of proximal tubule-derived OK cells

Cell biological approaches were usedto examine the location and function of the brush border (BB)Na⁺/H⁺ exchanger NHE3 in the opossum kidney(OK) polarized renal proximal tubule cell line. NHE3 epitope taggedwith the vesicular stomatitis virus glycoprotein epitope(NHE3V) was stably expressed and called OK-E3V cells. On thebasis of cell surface biotinylation studies, these cells had10-15% of total NHE3 on the BB. Intracellular NHE3V largelycolocalized with Rab11 and to a lesser extent with EEA1. The BBlocation of NHE3V was examined by confocal microscopy relative to thelectins wheat germ aggluttinin (WGA) and phytohemagluttin E (PHA-E), aswell 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 were diffusely distributed on the BB.Detergent extraction showed that total NHE3V was largely soluble inTriton X-100, whereas virtually all surface NHE3V was insoluble.Sucrose density gradient centrifugation demonstrated that total NHE3Vmigrated at the same size as ~400- and ~900-kDa standards, whereassurface NHE3V was enriched in the ~900-kDa form. Under basalconditions, NHE3 cycled between the cell surface and the recyclingpathway through a phosphatidylinositol (PI) 3-kinase-dependentmechanism. Measurements of surface and intracellular pH were obtainedby using FITC-WGA. Internalization of FITC-WGA occurred largely intothe juxtanuclear compartment that contained Rab11 and NHE3V. pH valueson the apical surface and in endosomes in the presence of the NHE3blocker, S3226, were elevated, showing that NHE3 functioned to acidifyboth compartments. In conclusion, NHE3V in OK cells exists in distinctdomains both in the center of the apical surface and in a juxtanuclearcompartment. In the BB fraction, NHE3 is largely in thedetergent-insoluble fraction in lipid rafts and/or in largeheterogenous complexes ranging from ~400 to ~900 kDa.