Active (9.6 S) and Inactive (21 S) Oligomers of NHE3 in Microdomains of the Renal Brush Border

Abstract

We have previously shown that Na ؉ -H ؉ exchanger isoform NHE3 exists as both 9.6 and 21 S (megalin-associated) oligomers in the renal brush border (1). To characterize the oligomeric forms of the renal brush border Na ؉ -H ؉ exchanger in more detail, we performed membrane fractionation studies. We found that similar amounts of NHE3 were present in microvilli and a nonmicrovillar membrane domain of high density (dense vesicles). Horseradish peroxidase-labeled endosomes were not prevalent in the dense membrane fraction. However, megalin, which localizes primarily to the intermicrovillar microdomain of the brush border, was enriched in the dense vesicles, implicating this microdomain as the likely source of these membranes. Immunolocalization of NHE3 confirmed that a major fraction of the transporter colocalized with megalin in the intermicrovillar region of the brush border. Immunoprecipitation studies demonstrated that in microvilli the majority of NHE3 was not bound to megalin, while in the dense vesicles most of the NHE3 coprecipitated with megalin. Moreover, sucrose velocity gradient centrifugation experiments revealed that most NHE3 in microvilli sedimented with an S value of 9.6, while the S value of NHE3 in dense vesicles was 21. Finally, we examined the functional state of NHE3 in both membrane fractions. As assayed by changes in acridine orange fluorescence, imposing an outwardly directed Na ؉ gradient caused generation of an inside acid pH gradient in the microvilli, indicating Na ؉ -H ؉ exchange activity, but not in the dense vesicles. Taken together, these data demonstrate that renal brush border NHE3 exists in two oligomeric states: a 9.6 S active form present in microvilli and a 21 S, megalin-associated, inactive form in the intermicrovillar microdomain of the apical plasma membrane. Thus, regulation of renal brush border Na ؉ -H ؉ exchange activity may be mediated by shifting the distribution between these forms of NHE3.In the kidney, the activity of the Na ϩ -H ϩ exchanger isoform NHE3, located on the apical microvillar membrane of the proximal tubule, plays a major role in mediating transepithelial bicarbonate and NaCl reabsorption (2-4). Numerous physiologic studies of brush border Na ϩ -H ϩ exchange have shown that this activity is regulated by such hormones as angiotensin II (5) and parathyroid hormone (6) as well as by systemic alterations in acid-base balance (2, 3). Several laboratories have presented evidence suggesting that NHE3 is regulated by posttranslational mechanisms that may include membrane trafficking between an intracellular compartment and the plasma membrane (7-9). Such models predict that NHE3 must be localized in a nonmicrovillar membrane compartment, which functions as a store of transporter, as well as on the microvillar membrane where NHE3 is active.We have recently reported an association between NHE3 and the putative scavenger receptor megalin in renal brush border membranes (1). Moreover, we found that renal brush border NHE3 exists in two states with distinct sediment...

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