Noel J, Roux D, Pouysségur J
Centre de Biochimie-CNRS, Université de Nice, France.
J Cell Sci. 1996 May;109 ( Pt 5):929-39. doi: 10.1242/jcs.109.5.929.
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.
钠/氢交换体（NHEs）是一类转运蛋白，它们以化学梯度为代价，在质膜上交换钠离子和质子离子。在高等真核生物中，这些转运蛋白以多种特殊的异构体形式存在。例如，广泛表达的NHE1是主要的pH调节系统，而上皮NHE3异构体则专门负责跨上皮钠离子转运。NHE1和NHE3可以通过HOE694特异性抑制剂进行很好的药理学区分，也可以通过特异性多克隆和单克隆抗体进行免疫区分。利用这些分子工具，我们研究了两种NHE异构体在培养的极化上皮细胞中的特异性稳态表达。OK细胞中的内源性NHE3或在OK细胞或MDCK细胞中瞬时转染的NHE3-VSVG显示该转运蛋白仅在顶端膜表达。NHE3的过表达不会导致其在基底外侧有任何溢出。通过NHE3活性的功能测量获得的这些结果与通过免疫荧光和共聚焦显微镜仅在顶端侧检测到该蛋白完全一致。相比之下，使用相同的细胞、相同的培养条件和相同的检测方法，我们在四种不同的极化上皮细胞系的两个特化膜上都清楚地检测到了NHE1。此外，对MDCK、OK和HT-29细胞的细胞表面蛋白进行生物素化，随后对NHE1进行免疫沉淀，结果显示该转运蛋白在极化上皮细胞的两侧均有表达。有趣的是，细胞表面表达与相应的NHE1活性密切相关。此外，通过荧光显微镜进行的免疫检测在质量上与上述结果一致。因此，我们得出结论，上皮性且更具特异性的NHE3异构体仅局限于上皮细胞的顶端侧。与之形成鲜明对比的是，内源性或异位表达的NHE1异构体都有能力在培养的极化细胞的顶端膜和基底外侧膜上表达。
