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

Abstract

Cell biological approaches were used to 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 tagged with the vesicular stomatitis virus glycoprotein epitope (NHE3V) was stably expressed and called OK-E3V cells. On the basis of cell surface biotinylation studies, these cells had 10-15% of total NHE3 on the BB. Intracellular NHE3V largely colocalized with Rab11 and to a lesser extent with EEA1. The BB location of NHE3V was examined by confocal microscopy relative to the lectins wheat germ aggluttinin (WGA) and phytohemagluttin E (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 were diffusely distributed on the BB. Detergent extraction showed that total NHE3V was largely soluble in Triton X-100, whereas virtually all surface NHE3V was insoluble. Sucrose density gradient centrifugation demonstrated that total NHE3V migrated at the same size as approximately 400- and approximately 900-kDa standards, whereas surface NHE3V was enriched in the approximately 900-kDa form. Under basal conditions, NHE3 cycled between the cell surface and the recycling pathway through a phosphatidylinositol (PI) 3-kinase-dependent mechanism. Measurements of surface and intracellular pH were obtained by 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 in OK cells exists in distinct domains both in the center of the apical surface and in a juxtanuclear compartment. In the BB fraction, NHE3 is largely in the detergent-insoluble fraction in lipid rafts and/or in large heterogenous complexes ranging from approximately 400 to approximately 900 kDa.

The lateral mobility of NHE3 on the apical membrane of renal epithelial OK cells is limited by the PDZ domain proteins NHERF1/2, but is dependent on an intact actin cytoskeleton as determined by FRAP

“…We previously showed that NHE3-EGFP localized in OK cells similarly to wild-type, being distributed in the apical membrane and in the juxtanuclear area. It was functionally inhibited by phosphoinositide 3-kinase inhibition (wortmannin), similarly to wildtype studies (Akhter et al, 2002). We have also reported that NHE3-EGFP stably expressed in PS120 cells behaves similarly to wild-type in terms of the percent amount on the surface, basal transport and stimulation by growth factors (Janecki et al, 2000a).…”

“…The mobile fraction (Mf) of apical NHE3-endosomes, including cellubrevin. NHE3 traffics between these two regions under basal conditions (D'Souza et al, 1998;Akhter et al, 2002). Most rapid regulation of NHE3 appears to involve changes in the amount of BB NHE3, which occurs from changes in the rates of NHE3 trafficking between the BB and the intracellular recycling compartment (Kurashima et al, 1998).…”

NHE3 Activity Is Dependent on Direct Phosphoinositide Binding at the N Terminus of Its Intracellular Cytosolic Region

“…Basal NHE3 activity was inhibited by exposure to the PI3K inhibitors, viz. LY594002 (31) and wortmannin (32,33) at low doses, and acutely stimulated NHE3 was reduced by the same inhibitors between 50 and 100% based on the cell type expressing NHE3 and the ligand studied (FGF in PS120 cells by 50% (32); lysophosphatidic acid in opossum kidney cells by 100%) (31). These previous studies of PI3K dependence of basal and acutely stimulated NHE3 activity plus the rapid stimulation of NHE3 activity when PI(3,4,5) 3 was added to the Table 1.…”

Regulation of Expression and Localisation of the Na+/H+ Exchanger (NHE) 3 and the NHE Regulatory Factor 2 in Baboon Placental Syncytiotrophoblast by Oestrogen

“…Moreover, it has been proposed that the trafficking of NHE3 to/from the cell membrane and the juxtanuclear compartment [24] represents a physiologic mechanism by which NHE3 controls Na + /H + exchange in epithelial cells of the kidney, colon, small intestine and gallbladder [25,26]. It also appears that trafficking of NHE3 can be enhanced by NHERF2, apparently via activation of phospholipase C [26].…”

“…Moreover, it has been proposed that the trafficking of NHE3 to/from the cell membrane and the juxtanuclear compartment [24] represents a physiologic mechanism by which NHE3 controls Na + /H + exchange in epithelial cells of the kidney, colon, small intestine and gallbladder [25,26]. It also appears that trafficking of NHE3 can be enhanced by NHERF2, apparently via activation of phospholipase C [26]. Although the specific physiologic role(s) of NHE3 and NHERF2 in syncytiotrophoblast intracellular function remains to be elucidated, based on our studies we propose that estrogen plays an important role in regulating expression of components of the NHE system within and consequently development and function of the primate placental syncytiotrophoblast.…”