THE EPITHELIAL SODIUM-HYDROGEN ANTIPORTER NA+ H+ EXCHANGER-3 ACCUMULATES AND IS FUNCTIONAL IN RECYCLING ENDOSOMES/

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 membrane s of renal and intestinal epithelial cells, where it contributes to Na Cl (re)absorption. NHE activity has been detected in endomembrane vesi cles 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 Chine se hamster ovary cells. To circumvent the confounding effects of endog enous exchangers, epitope-tagged constructs of NHE1 and NHE3 were stab ly expressed in antiport-deficient (AP-1) cells, While NHE1 was found almost exclusively in the surface membrane, NHE3 was also found intrac ellularly, accumulating in a juxtanuclear compartment, Confocal micros copy showed this compartment to be distinct from the Golgi, trans-Gels network, and lysosomes. Instead, NHE3 colocalized with transferrin re ceptors and with cellubrevin, markers of recycling endosomes. The acti vity of NHE3 in endomembranes was assessed by targeting pH-sensitive p robes to the recycling endosomes using a chimeric cellubrevin construc t with an accessible extracellular epitope. Fluorescence ratio imaging indicated that cellubrevin resides intracellularly in an acidic compa rtment, In AP-1 cells, endosomal acidification was unaffected by omiss ion 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 i ndicate that NHE3 is present and active in recycling endosomes. By rec ruiting NHE3 to the plasma membrane, modulation of vesicular traffic c ould contribute to the regulation of Na+ reabsorption across epithelia .