Active (9.6 S) and inactive (21 S) oligomers of NHE3 in microdomains of the renal brush border

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+ excha nger in more detail, we performed membrane fractionation studies. We found that similar amounts of NHE3 were present in microvilli and a non-microvill ar membrane domain of high density (dense vesicles), Horseradish peroxidase -labeled endosomes were not prevalent in the dense membrane fraction, Howev er, megalin, which localizes primarily to the intermicrovillar microdomain of the brush border, was enriched in the dense vesicles, implicating this m icrodomain as the likely source of these membranes. Immunolocalization of N HE3 confirmed that a major fraction of the transporter colocalized with meg alin in the intermicrovillar region of the brush border. Immunoprecipitatio n studies demonstrated that in microvilli the majority of NHE3 was not boun d to megalin, while in the dense vesicles most of the NHE3 coprecipitated w ith megalin, Moreover, sucrose velocity gradient centrifugation experiments revealed that most NHE3 in microvilli sedimented with an S value of 9,6, w hile 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+ gradie nt caused generation of an inside acid pH gradient in the microvilli, indic ating Na+-H+ exchange activity, but not in the dense vesicles. Taken togeth er, these data demonstrate that renal brush border NHE3 exists in two oligo meric states: a 9.6 S active form present in microvilli and a 21 S, megalin -associated, inactive form in the intermicrovillar microdomain of the apica l plasma membrane, Thus, regulation of renal brush border Na+-H+ exchange a ctivity may be mediated by shifting the distribution between these forms of NHE3.