title: Angiotensin II-dependent phosphorylation at Ser11/Ser18 and Ser938 shifts the E2 conformations of rat kidney Na+/K+-ATPase.
abstract: Kidney plasma membranes, which contain a single α-1 isoform of Na+/K+-ATPase, simultaneously contain two sub-conformations of E2P, differing in their rate of digoxin release in response to Na+ and ATP. Treating cells with Ang II (angiotensin II) somehow changes the conformation of both, because it differentially inhibits the rate of digoxin release. In the present study we tested whether Ang II regulates release by increasing phosphorylation at Ser11/Ser18 and Ser938. Opossum kidney cells co-expressing the AT1a receptor and either α-1.wild-type, α-1.S11A/S18A or α-1.S938A were treated with or without 10 nM Ang II for 5 min, increasing phosphorylation at the three sites. Na+/K+-ATPase was bound to digoxin-affinity columns in the presence of Na+, ATP and Mg2+. A solution containing 30 mM NaCl and 3 mM ATP eluted ~20% of bound untreated Na+/K+-ATPase (Population #1). Pre-treating cells with Ang II slowed the elution of Population #1 in α-1.wild-type and α-1.S938A, but not α-1.S11A/S18A cells. Another 50% of bound Na+/K+-ATPase (Population #2) was subsequently eluted in two phases by a solution containing 150 mM NaCl and 3 mM ATP. Ang II increased the initial rate and slowed the second phase in α-1.wild-type, but not α-1.S938A, cells. Thus Ang II changes the conformation of two forms of EP2 via differential phosphorylation.
authors.affiliation: Department of Physiology, Wayne State University, School of Medicine, Detroit, MI 48201, USA.
authors.forename: Katherine J
authors.initials: KJ
authors.lastname: Massey
authors.forename: Quanwen
authors.initials: Q
authors.lastname: Li
authors.forename: Noreen F
authors.initials: NF
authors.lastname: Rossi
authors.forename: Raymond R
authors.initials: RR
authors.lastname: Mattingly
authors.forename: Douglas R
authors.initials: DR
authors.lastname: Yingst
mesh_terms: D019943:Amino Acid Substitution; D000804:Angiotensin II / Q000737:chemistry* / Q000494:pharmacology / Q000502:physiology; D000818:Animals; D000906:Antibodies / Q000737:chemistry; D015153:Blotting, Western; D002460:Cell Line; D002846:Chromatography, Affinity; D004077:Digoxin / Q000737:chemistry; D007668:Kidney / Q000166:cytology / Q000201:enzymology; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009893:Opossums; D010766:Phosphorylation; D011485:Protein Binding; D011487:Protein Conformation; D011817:Rabbits; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012694:Serine / Q000737:chemistry*; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D000906:Antibodies; D011994:Recombinant Proteins; D000804:Angiotensin II; D012694:Serine; D004077:Digoxin; D000254:Sodium-Potassium-Exchanging ATPase