title: Association of the epithelial sodium channel with Apx and alpha-spectrin in A6 renal epithelial cells.
abstract: Recent molecular cloning of the epithelial sodium channel (ENaC) provides the opportunity to identify ENaC-associated proteins that function in regulating its cell surface expression and activity. We have examined whether ENaC is associated with Apx (apical protein Xenopus) and the spectrin-based membrane cytoskeleton in Xenopus A6 renal epithelial cells. We have also addressed whether Apx is required for the expression of amiloride-sensitive Na(+) currents by cloned ENaC. Sucrose density gradient centrifugation of A6 cell detergent extracts showed co-sedimentation of xENaC, alpha-spectrin, and Apx. Immunoblot analysis of proteins co-immunoprecipitating under high stringency conditions from peak Xenopus ENaC/Apx-containing gradient fractions indicate that ENaC, Apx, and alpha-spectrin are associated in a macromolecular complex. To examine whether Apx is required for the functional expression of ENaC, alphabetagamma mENaC cRNAs were coinjected into Xenopus oocytes with Apx sense or antisense oligodeoxynucleotides. The two-electrode voltage clamp technique showed there was a marked reduction in amiloride-sensitive current in oocytes coinjected with antisense oligonucleotides when to compared with oocytes coinjected with sense oligonucleotides. These studies indicate that ENaC is associated in a macromolecular complex with Apx and alpha-spectrin in A6 cells and suggest that Apx is required for the functional expression of ENaC in Xenopus epithelia.
authors.affiliation: Department of Medicine, University of Pennsylvania, Philadelphia, PA, USA.
authors.forename: J B
authors.initials: JB
authors.lastname: Zuckerman
authors.forename: X
authors.initials: X
authors.lastname: Chen
authors.forename: J D
authors.initials: JD
authors.lastname: Jacobs
authors.forename: B
authors.initials: B
authors.lastname: Hu
authors.forename: T R
authors.initials: TR
authors.lastname: Kleyman
authors.forename: P R
authors.initials: PR
authors.lastname: Smith
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity; D002460:Cell Line; D004847:Epithelial Cells / Q000378:metabolism; D053503:Epithelial Sodium Channels; D005786:Gene Expression Regulation / Q000187:drug effects; D007668:Kidney / Q000166:cytology / Q000378:metabolism*; D008969:Molecular Sequence Data; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D010446:Peptide Fragments / Q000276:immunology; D011233:Precipitin Tests; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D015222:Sodium Channels / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013049:Spectrin / Q000378:metabolism*; D029867:Xenopus Proteins; D014982:Xenopus laevis
chemical_list: D053503:Epithelial Sodium Channels; D016376:Oligonucleotides, Antisense; D010446:Peptide Fragments; D011994:Recombinant Proteins; C078824:Shroom1 protein, Xenopus; D015222:Sodium Channels; D029867:Xenopus Proteins; D013049:Spectrin