Functional reconstitution of the cardiac sarcoplasmic reticulum Ca2(+)-ATPase with phospholamban in phospholipid vesicles.
The Ca2(+)-ATPase in cardiac sarcoplasmic reticulum (SR) is under regulation by phospholamban, an oligomeric proteolipid. To determine the molecular mechanism by which phospholamban regulates the Ca2(+)-ATPase, a reconstitution system was developed, using a freeze-thaw sonication procedure. The best rates of Ca2+ uptake (700 nmol/min/mg reconstituted vesicles compared with 800 nmol/min/mg SR vesicles) were observed when cholate and phosphatidylcholine were used at a ratio of cholate/phosphatidylcholine/Ca2(+)-ATPase of 2:80:1. The EC50 values for Ca2+ were 0.05 microM for both Ca2+ uptake and Ca2(+)-ATPase activity in the reconstituted vesicles compared with 0.63 microM Ca2+ in native SR vesicles. Inclusion of phospholamban in the reconstituted vesicles was associated with a significant inhibition of the initial rates of Ca2+ uptake at pCa 6.0. However, phosphorylation of phospholamban by the catalytic subunit of the cAMP-dependent protein kinase reversed the inhibitory effect on the Ca2+ pump. Similar findings were observed when a peptide, corresponding to amino acids 1-25 of phospholamban, was used. These findings indicate that phospholamban is an inhibitor of the Ca2(+)-ATPase in cardiac SR and phosphorylation of phospholamban relieves this inhibition. The mechanism by which phospholamban inhibits the Ca2+ pump is unknown, but our findings with the synthetic peptide suggest that a direct interaction between the Ca2(+)-ATPase and the hydrophilic portion of phospholamban may be one of the mechanisms for regulation.
Kim
H W
HW
Department of Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, Ohio 45267-0575.
Steenaart
N A
NA
Ferguson
D G
DG
Kranias
E G
EG
D000818:Animals; D001693:Biological Transport, Active; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D002474:Cell-Free System; D004285:Dogs; D007150:Immunohistochemistry; D066298:In Vitro Techniques; D007425:Intracellular Membranes / Q000201:enzymology; D007700:Kinetics; D008081:Liposomes; D008854:Microscopy, Electron; D009206:Myocardium / Q000201:enzymology; D010446:Peptide Fragments / Q000494:pharmacology; D010743:Phospholipids; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D000099211:Phospholamban
D002118:Calcium; D002135:Calcium-Binding Proteins; D000252:Calcium-Transporting ATPases; D008081:Liposomes; D010446:Peptide Fragments; D010743:Phospholipids; D000099211:Phospholamban