The Epithelial Sodium-Hydrogen Antiporter Na+/H+ Exchanger 3 Accumulates and Is Functional in Recycling Endosomes

Abstract

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.

The NHX Family of Na+-H+ Exchangers in Caenorhabditis elegans

“…At least four of the NHX proteins (NHX-2, -4, -6, and -7) are targeted to the plasma membrane; the fact that the mammalian exchanger NHE3 can reside in intracellular vesicles that fuse to the plasma membrane upon cell signaling (35) suggests that even intracellular isoforms have the potential to mediate plasma membrane currents under appropriate conditions. In addition, the identification of a relatively large number of putative intracellular Na ϩ -H ϩ exchangers in C. elegans (five) compared with mammals (two to date) provides us with a unique opportunity to use the genetic and reverse genetic techniques that have been established in nematodes to dissect the role that these proteins play in organelle and vesicle function.…”

Traditional and emerging roles for the SLC9 Na+/H+ exchangers

“…Due to their distinct localization to specific types of organelles it is likely that intracellular NHEs play a critical part in the regulation of organellar pH. In support of this, overexpression or loss of intracellular NHEs alters organellar pH [113,35,130,80]. This concept is however challenged by studies where knock-down or loss of individual organelle-specific NHEs had no effect on pH [38,147,52].…”

Rho GTPases dictate the mobility of the Na/H exchanger NHE3 in epithelia: Role in apical retention and targeting

“…Experiments were performed at least 72 h after the monolayer had reached confluence. The cDNA constructs used to express GFP coupled to glycerophosphoinositides (GPIs) or to the FcIIa receptor have been described elsewhere (7,8).…”

“…Because the commonly used antiport-deficient lines (PS120 and AP-1) are fibroblastic, they lack the unique polarity and targeting determinants of differentiated epithelia. As a result, when expressed in such cells, NHE3 resides largely in endomembranes, and only a minor (Ϸ15%) fraction reaches the plasmalemma, where it is dispersed homogeneously (7). For these reasons, the determinants of apical targeting and retention of NHE3 remain unknown.…”