Angiotensin II-dependent phosphorylation at Ser11/Ser18 and Ser938 shifts the E2 conformations of rat kidney Na+/K+-ATPase

摘要

pKidney plasma membranes, which contain a single α-1 isoform of Nasup+/sup/Ksup+/sup-ATPase, simultaneously contain two sub-conformations of Esub2/subP, differing in their rate of digoxin release in response to Nasup+/sup 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 Sersup11/sup/Sersup18/sup and Sersup938/sup. Opossum kidney cells co-expressing the ATsub1a/sub 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. Nasup+/sup/Ksup+/sup-ATPase was bound to digoxin-affinity columns in the presence of Nasup+/sup, ATP and Mgsup2+/sup. A solution containing 30 mM NaCl and 3 mM ATP eluted ~20% of bound untreated Nasup+/sup/Ksup+/sup-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 Nasup+/sup/Ksup+/sup-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 EPsub2/sub via differential phosphorylation./p