D'Souza S, Garcia-Cabado A, Yu F, Teter K, Lukacs G, Skorecki K, Moore H P, Orlowski J, Grinstein S
Division of Cell Biology, Hospital for Sick Children, Toronto, Ontario, Canada.
J Biol Chem. 1998 Jan 23;273(4):2035-43. doi: 10.1074/jbc.273.4.2035.
Na+/H+ exchangers (NHEs) mediate electroneutral exchange of Na+ for H+ and thereby play a central role in pH regulation and Na+ homeostasis. NHE3, the predominant epithelial isoform, is found in apical membranes of renal and intestinal epithelial cells, where it contributes to NaCl (re)absorption. NHE activity has been detected in endomembrane vesicles of epithelial cells, but the precise compartment involved and its functional role have not been defined. Many aspects of the targeting machinery that defines the compartmentation and polarity of epithelia are also functional in nonepithelial cells. We therefore compared the targeting of NHE1, the basolateral isoform, with that of NHE3 in Chinese hamster ovary cells. To circumvent the confounding effects of endogenous exchangers, epitope-tagged constructs of NHE1 and NHE3 were stably expressed in antiport-deficient (AP-1) cells. While NHE1 was found almost exclusively in the surface membrane, NHE3 was also found intracellularly, accumulating in a juxtanuclear compartment. Confocal microscopy showed this compartment to be distinct from the Golgi, trans-Golgi network, and lysosomes. Instead, NHE3 colocalized with transferrin receptors and with cellubrevin, markers of recycling endosomes. The activity of NHE3 in endomembranes was assessed by targeting pH-sensitive probes to the recycling endosomes using a chimeric cellubrevin construct with an accessible extracellular epitope. Fluorescence ratio imaging indicated that cellubrevin resides intracellularly in an acidic compartment. In AP-1 cells, endosomal acidification was unaffected by omission of Na+ but was dissipated entirely by concanamycin, a blocker of H(+)-ATPases. In contrast, the cellubrevin compartment was more acidic in NHE3 transfectants, and the acidification was only partially reduced by concanamycin. Moreover, removal of extracellular Na+ resulted in a significant alkalization of the endocytic compartment. These results indicate that NHE3 is present and active in recycling endosomes. By recruiting NHE3 to the plasma membrane, modulation of vesicular traffic could contribute to the regulation of Na+ reabsorption across epithelia.
钠氢交换体（NHEs）介导Na⁺与H⁺的电中性交换，从而在pH调节和Na⁺稳态中发挥核心作用。NHE3是主要的上皮亚型，存在于肾和肠上皮细胞的顶端膜中，在那里它有助于NaCl的（再）吸收。已在上皮细胞的内膜囊泡中检测到NHE活性，但所涉及的精确区室及其功能作用尚未明确。定义上皮细胞区室化和极性的靶向机制的许多方面在非上皮细胞中也起作用。因此，我们在中国仓鼠卵巢细胞中比较了基底外侧亚型NHE1和NHE3的靶向情况。为了规避内源性交换体的混杂影响，NHE1和NHE3的表位标记构建体在反向转运缺陷（AP-1）细胞中稳定表达。虽然NHE1几乎只存在于表面膜中，但NHE3也存在于细胞内，聚集在近核区室中。共聚焦显微镜显示该区室与高尔基体、反式高尔基体网络和溶酶体不同。相反，NHE3与转铁蛋白受体以及细胞ubrevin共定位，后者是再循环内体的标志物。通过使用具有可及细胞外表位的嵌合细胞ubrevin构建体将pH敏感探针靶向再循环内体，评估了NHE3在内膜中的活性。荧光比率成像表明细胞ubrevin存在于细胞内的酸性区室中。在AP-1细胞中，内体酸化不受Na⁺缺失的影响，但完全被H⁺-ATP酶阻滞剂 concanamycin消除。相反，在NHE3转染细胞中，细胞ubrevin区室酸性更强，并且酸化仅被concanamycin部分降低。此外，去除细胞外Na⁺导致内吞区室显著碱化。这些结果表明NHE3存在于再循环内体中并具有活性。通过将NHE3募集到质膜，囊泡运输的调节可能有助于调节上皮细胞对Na⁺的重吸收。
