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• W2024336491 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 (1Biemesderfer D. Nagy T. DeGray B. Aronson P.S. J. Biol. Chem. 1999; 274: 17518-17524Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). 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. 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 (1Biemesderfer D. Nagy T. DeGray B. Aronson P.S. J. Biol. Chem. 1999; 274: 17518-17524Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). 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. monoclonal antibody horseradish peroxidase N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine paraformaldehyde-lysine-periodate Tris-buffered saline 4-morpholineethanesulfonic acid In the kidney, the activity of the Na+-H+exchanger isoform NHE3, located on the apical microvillar membrane of the proximal tubule, plays a major role in mediating transepithelial bicarbonate and NaCl reabsorption (2Ambuhl P.M. Amemiya M. Danczkay M. Lotscher M. Kaissling B. Moe O.W. Presig P.A. Alpern R.J. Am. J. Physiol. 1996; 271: F917-F925PubMed Google Scholar, 3Wu M.S. Biemesderfer D. Giebisch G. Aronson P.S. J. Biol. Chem. 1996; 271: 32749-32752Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 4Aronson P.S. Giebisch G. Am. J. Physiol. 1997; 273: F179-F192Crossref PubMed Google Scholar). Numerous physiologic studies of brush border Na+-H+ exchange have shown that this activity is regulated by such hormones as angiotensin II (5Wang T. Chan Y.L. J. Pharmacol. Exp. Ther. 1990; 252: 689-695PubMed Google Scholar) and parathyroid hormone (6Iino Y. Burg M.B. Am. J. Physiol. 1979; 236: F387-F391PubMed Google Scholar) as well as by systemic alterations in acid-base balance (2Ambuhl P.M. Amemiya M. Danczkay M. Lotscher M. Kaissling B. Moe O.W. Presig P.A. Alpern R.J. Am. J. Physiol. 1996; 271: F917-F925PubMed Google Scholar, 3Wu M.S. Biemesderfer D. Giebisch G. Aronson P.S. J. Biol. Chem. 1996; 271: 32749-32752Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). 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 (7Block R.D. Zikos D. Fisher K.A. Sukowski E.J. Cargoe E.J. Peterson D.R. Contrib. Nephrol. 1993; 101: 19-25Crossref PubMed Google Scholar, 8Zhang Y. Norian J.M. Magyar C.E. Holstein-Rathlou N.H. Mircheff A.K. McDonough A.A. Am. J. Physiol. 1999; 276: F711-F719Crossref PubMed Google Scholar, 9Yang X. Amemiya M. Moe O.W. Presig P.A. Alpern R.J. J. Am. Soc. Nephrol. 1999; 10 (abstr.): 12Google Scholar). 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 (1Biemesderfer D. Nagy T. DeGray B. Aronson P.S. J. Biol. Chem. 1999; 274: 17518-17524Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). Moreover, we found that renal brush border NHE3 exists in two states with distinct sedimentation coefficients, a 9.6 S megalin-free form and a 21 S megalin-bound form (1Biemesderfer D. Nagy T. DeGray B. Aronson P.S. J. Biol. Chem. 1999; 274: 17518-17524Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). The purpose of the present study was to use membrane fractionation methods to determine whether these two oligomeric forms of NHE3 are expressed in distinct microdomains of the renal brush border and, if so, to compare the functional activity of NHE3 in these microdomains. Our findings are consistent with a model in which the 9.6 S form of NHE3 is present in the microvillar membrane and is active, whereas the 21 S megalin-associated form is concentrated in the intermicrovillar microdomain of the brush border and is inactive. Thus, regulation of renal brush border Na+-H+exchange activity may be mediated by shifting the distribution between these forms of NHE3.DISCUSSIONThis study describes the localization of two oligomeric forms of NHE3 to distinct microdomains of the renal brush border. Using density centrifugation combined with specific brush border antibodies, we have shown that a 9.6 S oligomer of NHE3 is predominately expressed on microvilli, while a 21 S, megalin-associated form is concentrated in the intermicrovillar region of the brush border. As shown by Rodmanet al. (32Rodman J.S. Seidman L. Farquhar M.G. J. Cell Biol. 1986; 102: 77-87Crossref PubMed Scopus (57) Google Scholar), this is a distinct microdomain of the apical plasma membrane of the proximal tubule. In addition, transport studies indicate that NHE3 is active in microvilli but inactive in intermicrovillar membranes.The mAb to NHE3 (2B9) that was used in this study is specific to a region within the C terminus of the transporter that lies between amino acids 702 and 756 (10Biemesderfer D. Rutherford P.A. Nagy T. Pizzonia J.H. Abu-Alfa A.K. Aronson P.S. Am. J. Physiol. 1997; 273: F289-F299Crossref PubMed Google Scholar). This region of NHE3 is blocked from antibody binding when the transporter is complexed with megalin (1Biemesderfer D. Nagy T. DeGray B. Aronson P.S. J. Biol. Chem. 1999; 274: 17518-17524Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). The fact that epitopes within the C-terminal region of NHE3 are blocked when NHE3 is complexed with megalin raises questions regarding the interpretation of previous immunocytochemical studies using antibodies made to the C terminus of NHE3. Most (10Biemesderfer D. Rutherford P.A. Nagy T. Pizzonia J.H. Abu-Alfa A.K. Aronson P.S. Am. J. Physiol. 1997; 273: F289-F299Crossref PubMed Google Scholar, 33Biemesderfer D. Pizzonia J. Abu-Alfa A. Exner M. Reilly R. Igarashi P. Aronson P.S. Am. J. Physiol. 1993; 265: F736-F742PubMed Google Scholar, 34Amemiya M. Loffiing J. Lotscher M. Kaissling B. Alpern R.J. Moe O.W. Kidney Int. 1995; 48: 1206-1215Abstract Full Text PDF PubMed Scopus (348) Google Scholar) but not all (12Kim G.-H. Ecelbarger C. Knepper M.A. Packer R.K. J. Am. Soc. Nephrol. 1999; 10: 935-942PubMed Google Scholar) of these studies localized NHE3 largely to the microvillar membrane. In fact, when used for immunoelectron microscopy, our mAbs failed to label the intermicrovillar microdomain of the renal brush border (10Biemesderfer D. Rutherford P.A. Nagy T. Pizzonia J.H. Abu-Alfa A.K. Aronson P.S. Am. J. Physiol. 1997; 273: F289-F299Crossref PubMed Google Scholar). We hypothesized that the apparent discrepancy between previous immunocytochemical studies and the present membrane fractionation study, showing large amounts of NHE3 in intermicrovillar membranes, may have resulted from the masking of C-terminal epitopes on NHE3 when the transporter is complexed with megalin. Our present immunocytochemical studies using antigen retrieval show this to be the case. When kidney sections were denatured during the antigen retrieval procedure, we visualized a large pool of NHE3 that colocalized with megalin in the intermicrovillar region of the brush border, consistent with our membrane fractionation data showing a major pool of NHE3 in megalin-rich, nonmicrovillar (and nonendosomal) dense membranes. Immunocytochemical localization of this pool of NHE3 was not an artifact of the antigen retrieval procedure, since similar staining was found without antigen retrieval by use of a polyclonal anti-NHE3 antibody raised to a peptide encompassing the C-terminal 22 amino acids of NHE3 (12Kim G.-H. Ecelbarger C. Knepper M.A. Packer R.K. J. Am. Soc. Nephrol. 1999; 10: 935-942PubMed Google Scholar).Previous studies have localized Na+/H+ exchange activity (35Sabolic I. Brown D. Am. J. Physiol. 1990; 258: F1245-F1253PubMed Google Scholar) and NHE3 protein (10Biemesderfer D. Rutherford P.A. Nagy T. Pizzonia J.H. Abu-Alfa A.K. Aronson P.S. Am. J. Physiol. 1997; 273: F289-F299Crossref PubMed Google Scholar) within endosomes in the proximal tubule. In contrast, the data presented here indicate that the membranes enriched in the NHE3-megalin complex are derived from the intermicrovillar region of the brush border (apical plasma membrane) and not from an endosomal compartment. Since the present study did not directly examine the NHE3 protein present in endosomes, the relationship between endosomal NHE3 and that found in the intermicrovillar microdomain of the brush border remains unclear. However, because the NHE3 antibodies that we used in a previous study to detect NHE3 in endosomes (10Biemesderfer D. Rutherford P.A. Nagy T. Pizzonia J.H. Abu-Alfa A.K. Aronson P.S. Am. J. Physiol. 1997; 273: F289-F299Crossref PubMed Google Scholar) do not react with the NHE3-megalin complex (1Biemesderfer D. Nagy T. DeGray B. Aronson P.S. J. Biol. Chem. 1999; 274: 17518-17524Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar), it seems likely that endosomal staining with these mAbs was due to the presence of the 9.6 S form of NHE3 in the endosomes and not the NHE3-megalin complex.Our finding that the 21 S megalin-associated form of NHE3 concentrated in the intermicrovillar membrane is inactive raises the possibility that association with megalin may play a role in regulating NHE3 activity in response to physiological stimuli. For example, it is possible that the association of NHE3 with megalin causes inactivation of transport activity. However, it is known that phosphorylation of NHE3 by protein kinase A causes inhibition of NHE3 activity (36Kurashima K., Yu, F.H. Cabado A.G. Szabo E.Z. Grinstein S. Orlowski J. J. Biol. Chem. 1997; 272: 28672-28679Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 37Cabado A.G., Yu, F.H. Kapus A. Grinstein S. Orlowski J. J. Biol. Chem. 1996; 271: 3590-3599Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). It is possible that phosphorylation of NHE3 causes both its inactivation and its association with megalin. In addition, endocytosis of NHE3 in response to protein kinase A-induced phosphorylation has been described in cultured cell models (38Chow C.W. Khurana S. Woodside M. Grinstein S. Orlowski J. J. Biol. Chem. 1999; 274: 37551-37558Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 39Fan L. Wederkehr M.R. Collazo R. Wang H. Crowder L.A. Moe O.W. J. Biol. Chem. 1999; 274: 11289-11295Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). It is therefore also possible that phosphorylation of NHE3 leads to association with megalin and sequestration in the coated pits as a prelude to endocytosis in proximal tubule cells.In conclusion, we have demonstrated 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. Future studies will be needed to examine whether 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+-H+exchanger isoform NHE3, located on the apical microvillar membrane of the proximal tubule, plays a major role in mediating transepithelial bicarbonate and NaCl reabsorption (2Ambuhl P.M. Amemiya M. Danczkay M. Lotscher M. Kaissling B. Moe O.W. Presig P.A. Alpern R.J. Am. J. Physiol. 1996; 271: F917-F925PubMed Google Scholar, 3Wu M.S. Biemesderfer D. Giebisch G. Aronson P.S. J. Biol. Chem. 1996; 271: 32749-32752Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 4Aronson P.S. Giebisch G. Am. J. Physiol. 1997; 273: F179-F192Crossref PubMed Google Scholar). Numerous physiologic studies of brush border Na+-H+ exchange have shown that this activity is regulated by such hormones as angiotensin II (5Wang T. Chan Y.L. J. Pharmacol. Exp. Ther. 1990; 252: 689-695PubMed Google Scholar) and parathyroid hormone (6Iino Y. Burg M.B. Am. J. Physiol. 1979; 236: F387-F391PubMed Google Scholar) as well as by systemic alterations in acid-base balance (2Ambuhl P.M. Amemiya M. Danczkay M. Lotscher M. Kaissling B. Moe O.W. Presig P.A. Alpern R.J. Am. J. Physiol. 1996; 271: F917-F925PubMed Google Scholar, 3Wu M.S. Biemesderfer D. Giebisch G. Aronson P.S. J. Biol. Chem. 1996; 271: 32749-32752Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). 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 (7Block R.D. Zikos D. Fisher K.A. Sukowski E.J. Cargoe E.J. Peterson D.R. Contrib. Nephrol. 1993; 101: 19-25Crossref PubMed Google Scholar, 8Zhang Y. Norian J.M. Magyar C.E. Holstein-Rathlou N.H. Mircheff A.K. McDonough A.A. Am. J. Physiol. 1999; 276: F711-F719Crossref PubMed Google Scholar, 9Yang X. Amemiya M. Moe O.W. Presig P.A. Alpern R.J. J. Am. Soc. Nephrol. 1999; 10 (abstr.): 12Google Scholar). 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 (1Biemesderfer D. Nagy T. DeGray B. Aronson P.S. J. Biol. Chem. 1999; 274: 17518-17524Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). Moreover, we found that renal brush border NHE3 exists in two states with distinct sedimentation coefficients, a 9.6 S megalin-free form and a 21 S megalin-bound form (1Biemesderfer D. Nagy T. DeGray B. Aronson P.S. J. Biol. Chem. 1999; 274: 17518-17524Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). The purpose of the present study was to use membrane fractionation methods to determine whether these two oligomeric forms of NHE3 are expressed in distinct microdomains of the renal brush border and, if so, to compare the functional activity of NHE3 in these microdomains. Our findings are consistent with a model in which the 9.6 S form of NHE3 is present in the microvillar membrane and is active, whereas the 21 S megalin-associated form is concentrated in the intermicrovillar microdomain of the brush border and is inactive. Thus, regulation of renal brush border Na+-H+exchange activity may be mediated by shifting the distribution between these forms of NHE3. DISCUSSIONThis study describes the localization of two oligomeric forms of NHE3 to distinct microdomains of the renal brush border. Using density centrifugation combined with specific brush border antibodies, we have shown that a 9.6 S oligomer of NHE3 is predominately expressed on microvilli, while a 21 S, megalin-associated form is concentrated in the intermicrovillar region of the brush border. As shown by Rodmanet al. (32Rodman J.S. Seidman L. Farquhar M.G. J. Cell Biol. 1986; 102: 77-87Crossref PubMed Scopus (57) Google Scholar), this is a distinct microdomain of the apical plasma membrane of the proximal tubule. In addition, transport studies indicate that NHE3 is active in microvilli but inactive in intermicrovillar membranes.The mAb to NHE3 (2B9) that was used in this study is specific to a region within the C terminus of the transporter that lies between amino acids 702 and 756 (10Biemesderfer D. Rutherford P.A. Nagy T. Pizzonia J.H. Abu-Alfa A.K. Aronson P.S. Am. J. Physiol. 1997; 273: F289-F299Crossref PubMed Google Scholar). This region of NHE3 is blocked from antibody binding when the transporter is complexed with megalin (1Biemesderfer D. Nagy T. DeGray B. Aronson P.S. J. Biol. Chem. 1999; 274: 17518-17524Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). The fact that epitopes within the C-terminal region of NHE3 are blocked when NHE3 is complexed with megalin raises questions regarding the interpretation of previous immunocytochemical studies using antibodies made to the C terminus of NHE3. Most (10Biemesderfer D. Rutherford P.A. Nagy T. Pizzonia J.H. Abu-Alfa A.K. Aronson P.S. Am. J. Physiol. 1997; 273: F289-F299Crossref PubMed Google Scholar, 33Biemesderfer D. Pizzonia J. Abu-Alfa A. Exner M. Reilly R. Igarashi P. Aronson P.S. Am. J. Physiol. 1993; 265: F736-F742PubMed Google Scholar, 34Amemiya M. Loffiing J. Lotscher M. Kaissling B. Alpern R.J. Moe O.W. Kidney Int. 1995; 48: 1206-1215Abstract Full Text PDF PubMed Scopus (348) Google Scholar) but not all (12Kim G.-H. Ecelbarger C. Knepper M.A. Packer R.K. J. Am. Soc. Nephrol. 1999; 10: 935-942PubMed Google Scholar) of these studies localized NHE3 largely to the microvillar membrane. In fact, when used for immunoelectron microscopy, our mAbs failed to label the intermicrovillar microdomain of the renal brush border (10Biemesderfer D. Rutherford P.A. Nagy T. Pizzonia J.H. Abu-Alfa A.K. Aronson P.S. Am. J. Physiol. 1997; 273: F289-F299Crossref PubMed Google Scholar). We hypothesized that the apparent discrepancy between previous immunocytochemical studies and the present membrane fractionation study, showing large amounts of NHE3 in intermicrovillar membranes, may have resulted from the masking of C-terminal epitopes on NHE3 when the transporter is complexed with megalin. Our present immunocytochemical studies using antigen retrieval show this to be the case. When kidney sections were denatured during the antigen retrieval procedure, we visualized a large pool of NHE3 that colocalized with megalin in the intermicrovillar region of the brush border, consistent with our membrane fractionation data showing a major pool of NHE3 in megalin-rich, nonmicrovillar (and nonendosomal) dense membranes. Immunocytochemical localization of this pool of NHE3 was not an artifact of the antigen retrieval procedure, since similar staining was found without antigen retrieval by use of a polyclonal anti-NHE3 antibody raised to a peptide encompassing the C-terminal 22 amino acids of NHE3 (12Kim G.-H. Ecelbarger C. Knepper M.A. Packer R.K. J. Am. Soc. Nephrol. 1999; 10: 935-942PubMed Google Scholar).Previous studies have localized Na+/H+ exchange activity (35Sabolic I. Brown D. Am. J. Physiol. 1990; 258: F1245-F1253PubMed Google Scholar) and NHE3 protein (10Biemesderfer D. Rutherford P.A. Nagy T. Pizzonia J.H. Abu-Alfa A.K. Aronson P.S. Am. J. Physiol. 1997; 273: F289-F299Crossref PubMed Google Scholar) within endosomes in the proximal tubule. In contrast, the data presented here indicate that the membranes enriched in the NHE3-megalin complex are derived from the intermicrovillar region of the brush border (apical plasma membrane) and not from an endosomal compartment. Since the present study did not directly examine the NHE3 protein present in endosomes, the relationship between endosomal NHE3 and that found in the intermicrovillar microdomain of the brush border remains unclear. However, because the NHE3 antibodies that we used in a previous study to detect NHE3 in endosomes (10Biemesderfer D. Rutherford P.A. Nagy T. Pizzonia J.H. Abu-Alfa A.K. Aronson P.S. Am. J. Physiol. 1997; 273: F289-F299Crossref PubMed Google Scholar) do not react with the NHE3-megalin complex (1Biemesderfer D. Nagy T. DeGray B. Aronson P.S. J. Biol. Chem. 1999; 274: 17518-17524Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar), it seems likely that endosomal staining with these mAbs was due to the presence of the 9.6 S form of NHE3 in the endosomes and not the NHE3-megalin complex.Our finding that the 21 S megalin-associated form of NHE3 concentrated in the intermicrovillar membrane is inactive raises the possibility that association with megalin may play a role in regulating NHE3 activity in response to physiological stimuli. For example, it is possible that the association of NHE3 with megalin causes inactivation of transport activity. However, it is known that phosphorylation of NHE3 by protein kinase A causes inhibition of NHE3 activity (36Kurashima K., Yu, F.H. Cabado A.G. Szabo E.Z. Grinstein S. Orlowski J. J. Biol. Chem. 1997; 272: 28672-28679Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 37Cabado A.G., Yu, F.H. Kapus A. Grinstein S. Orlowski J. J. Biol. Chem. 1996; 271: 3590-3599Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). It is possible that phosphorylation of NHE3 causes both its inactivation and its association with megalin. In addition, endocytosis of NHE3 in response to protein kinase A-induced phosphorylation has been described in cultured cell models (38Chow C.W. Khurana S. Woodside M. Grinstein S. Orlowski J. J. Biol. Chem. 1999; 274: 37551-37558Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 39Fan L. Wederkehr M.R. Collazo R. Wang H. Crowder L.A. Moe O.W. J. Biol. Chem. 1999; 274: 11289-11295Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). It is therefore also possible that phosphorylation of NHE3 leads to association with megalin and sequestration in the coated pits as a prelude to endocytosis in proximal tubule cells.In conclusion, we have demonstrated 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. Future studies will be needed to examine whether regulation of renal brush border Na+-H+exchange activity may be mediated by shifting the distribution between these forms of NHE3. This study describes the localization of two oligomeric forms of NHE3 to distinct microdomains of the renal brush border. Using density centrifugation combined with specific brush border antibodies, we have shown that a 9.6 S oligomer of NHE3 is predominately expressed on microvilli, while a 21 S, megalin-associated form is concentrated in the intermicrovillar region of the brush border. As shown by Rodmanet al. (32Rodman J.S. Seidman L. Farquhar M.G. J. Cell Biol. 1986; 102: 77-87Crossref PubMed Scopus (57) Google Scholar), this is a distinct microdomain of the apical plasma membrane of the proximal tubule. In addition, transport studies indicate that NHE3 is active in microvilli but inactive in intermicrovillar membranes. The mAb to NHE3 (2B9) that was used in this study is specific to a region within the C terminus of the transporter that lies between amino acids 702 and 756 (10Biemesderfer D. Rutherford P.A. Nagy T. Pizzonia J.H. Abu-Alfa A.K. Aronson P.S. Am. J. Physiol. 1997; 273: F289-F299Crossref PubMed Google Scholar). This region of NHE3 is blocked from antibody binding when the transporter is complexed with megalin (1Biemesderfer D. Nagy T. DeGray B. Aronson P.S. J. Biol. Chem. 1999; 274: 17518-17524Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). The fact that epitopes within the C-terminal region of NHE3 are blocked when NHE3 is complexed with megalin raises questions regarding the interpretation of previous immunocytochemical studies using antibodies made to the C terminus of NHE3. Most (10Biemesderfer D. Rutherford P.A. Nagy T. Pizzonia J.H. Abu-Alfa A.K. Aronson P.S. Am. J. Physiol. 1997; 273: F289-F299Crossref PubMed Google Scholar, 33Biemesderfer D. Pizzonia J. Abu-Alfa A. Exner M. Reilly R. Igarashi P. Aronson P.S. Am. J. Physiol. 1993; 265: F736-F742PubMed Google Scholar, 34Amemiya M. Loffiing J. Lotscher M. Kaissling B. Alpern R.J. Moe O.W. Kidney Int. 1995; 48: 1206-1215Abstract Full Text PDF PubMed Scopus (348) Google Scholar) but not all (12Kim G.-H. Ecelbarger C. Knepper M.A. Packer R.K. J. Am. Soc. Nephrol. 1999; 10: 935-942PubMed Google Scholar) of these studies localized NHE3 largely to the microvillar membrane. In fact, when used for immunoelectron microscopy, our mAbs failed to label the intermicrovillar microdomain of the renal brush border (10Biemesderfer D. Rutherford P.A. Nagy T. Pizzonia J.H. Abu-Alfa A.K. Aronson P.S. Am. J. Physiol. 1997; 273: F289-F299Crossref PubMed Google Scholar). We hypothesized that the apparent discrepancy between previous immunocytochemical studies and the present membrane fractionation study, showing large amounts of NHE3 in intermicrovillar membranes, may have resulted from the masking of C-terminal epitopes on NHE3 when the transporter is complexed with megalin. Our present immunocytochemical studies using antigen retrieval show this to be the case. When kidney sections were denatured during the antigen retrieval procedure, we visualized a large pool of NHE3 that colocalized with megalin in the intermicrovillar region of the brush border, consistent with our membrane fractionation data showing a major pool of NHE3 in megalin-rich, nonmicrovillar (and nonendosomal) dense membranes. Immunocytochemical localization of this pool of NHE3 was not an artifact of the antigen retrieval procedure, since similar staining was found without antigen retrieval by use of a polyclonal anti-NHE3 antibody raised to a peptide encompassing the C-terminal 22 amino acids of NHE3 (12Kim G.-H. Ecelbarger C. Knepper M.A. Packer R.K. J. Am. Soc. Nephrol. 1999; 10: 935-942PubMed Google Scholar). Previous studies have localized Na+/H+ exchange activity (35Sabolic I. Brown D. Am. J. Physiol. 1990; 258: F1245-F1253PubMed Google Scholar) and NHE3 protein (10Biemesderfer D. Rutherford P.A. Nagy T. Pizzonia J.H. Abu-Alfa A.K. Aronson P.S. Am. J. Physiol. 1997; 273: F289-F299Crossref PubMed Google Scholar) within endosomes in the proximal tubule. In contrast, the data presented here indicate that the membranes enriched in the NHE3-megalin complex are derived from the intermicrovillar region of the brush border (apical plasma membrane) and not from an endosomal compartment. Since the present study did not directly examine the NHE3 protein present in endosomes, the relationship between endosomal NHE3 and that found in the intermicrovillar microdomain of the brush border remains unclear. However, because the NHE3 antibodies that we used in a previous study to detect NHE3 in endosomes (10Biemesderfer D. Rutherford P.A. Nagy T. Pizzonia J.H. Abu-Alfa A.K. Aronson P.S. Am. J. Physiol. 1997; 273: F289-F299Crossref PubMed Google Scholar) do not react with the NHE3-megalin complex (1Biemesderfer D. Nagy T. DeGray B. Aronson P.S. J. Biol. Chem. 1999; 274: 17518-17524Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar), it seems likely that endosomal staining with these mAbs was due to the presence of the 9.6 S form of NHE3 in the endosomes and not the NHE3-megalin complex. Our finding that the 21 S megalin-associated form of NHE3 concentrated in the intermicrovillar membrane is inactive raises the possibility that association with megalin may play a role in regulating NHE3 activity in response to physiological stimuli. For example, it is possible that the association of NHE3 with megalin causes inactivation of transport activity. However, it is known that phosphorylation of NHE3 by protein kinase A causes inhibition of NHE3 activity (36Kurashima K., Yu, F.H. Cabado A.G. Szabo E.Z. Grinstein S. Orlowski J. J. Biol. Chem. 1997; 272: 28672-28679Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 37Cabado A.G., Yu, F.H. Kapus A. Grinstein S. Orlowski J. J. Biol. Chem. 1996; 271: 3590-3599Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). It is possible that phosphorylation of NHE3 causes both its inactivation and its association with megalin. In addition, endocytosis of NHE3 in response to protein kinase A-induced phosphorylation has been described in cultured cell models (38Chow C.W. Khurana S. Woodside M. Grinstein S. Orlowski J. J. Biol. Chem. 1999; 274: 37551-37558Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 39Fan L. Wederkehr M.R. Collazo R. Wang H. Crowder L.A. Moe O.W. J. Biol. Chem. 1999; 274: 11289-11295Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). It is therefore also possible that phosphorylation of NHE3 leads to association with megalin and sequestration in the coated pits as a prelude to endocytosis in proximal tubule cells. In conclusion, we have demonstrated 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. Future studies will be needed to examine whether regulation of renal brush border Na+-H+exchange activity may be mediated by shifting the distribution between these forms of NHE3." @default.
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