title: Differential localization of Na+/H+ exchanger isoforms (NHE1 and NHE3) in polarized epithelial cell lines.
abstract: Na+/H+ exchangers (NHEs) are transporters that exchange sodium and proton ions across the plasma membrane at the expense of their chemical gradient. In higher eukaryotes these transporters exist as multiple specialized isoforms. For example, NHE1, the ubiquitously expressed form is a major pH-regulating system whereas the epithelial NHE3 isoform is specialized in transepithelial Na+ transport. NHE1 and NHE3 can be very well distinguished pharmacologically with the HOE694 specific inhibitor and immunologically with specific polyclonal and monoclonal antibodies. With these molecular tools we investigated the specific steady state expression of the two NHE isoforms in polarized epithelial cells in culture. Endogenous NHE3 in OK cells or NHE3-VSVG transfected in either OK or MDCK cells showed an exclusive expression of the transporter at the apical membrane. Overexpression of NHE3 did not result in any spill over on the basal lateral side. These results obtained by functional measurement of NHE3 activity were fully consistent with its detection only at the apical side by immunofluorescence and confocal microscopy. By contrast, using the same cells, the same culture conditions and the same detection methods, we clearly detected NHE1 at both specialized membranes of four different polarized epithelial cell lines. Furthermore, biotinylation of cell surface proteins of MDCK, OK and HT-29 cells followed by immunoprecipitation of NHE1 revealed expression of the transporter at both sides of the polarized epithelial cells. Interestingly, the cell surface expression correlated well with the corresponding NHE1 activities. In addition, immunodetection by fluorescence microscopy was found to be qualitatively consistent with the above-reported results. We therefore conclude that the epithelial and more specialized NHE3 isoform is exclusively restricted to the apical side of epithelial cells. In marked contrast, both endogenous or ectopically expressed NHE1 isoform, have the capacity to be expressed in both the apical and basal lateral membranes of polarized cells in cultures.
authors: Centre de Biochimie-CNRS, Université de Nice, France.
authors: J
authors: J
authors: Noel
authors: D
authors: D
authors: Roux
authors: J
authors: J
authors: Pouysségur
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000502:physiology; D016764:Cell Polarity; D006224:Cricetinae; D003412:Cricetulus; D004285:Dogs; D004847:Epithelial Cells; D008969:Molecular Sequence Data; D009893:Opossums; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers / Q000032:analysis*; D014162:Transfection
chemical_list: D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers; C069139:growth factor-activatable Na-H exchanger NHE-1