High-level coexpression of the canine cardiac calcium pump and phospholamban in Sf21 insect cells.
Phospholamban is a pentameric transmembrane phosphoprotein that regulates the activity of the Ca(2+)-transporting ATPase (SERCA2a) in cardiac sarcoplasmic reticulum. To better understand the structure and function of phospholamban and its mode of regulation of the ATPase, phospholamban and SERCA2a were coexpressed at high levels in Sf21 insect cells using the baculovirus expression system. SERCA2a was expressed as a functionally active Ca2+ pump, accounting for > or = 20% of the total protein in Sf21 cell microsomes. Wild-type phospholamban, as well as phospholamban with different point mutations in the transmembrane region, inhibited both Ca2+ transport and ATP hydrolysis by the recombinant Ca2+ pump. The inhibition of SERCA2a activity was reversed by an anti-phospholamban monoclonal antibody. The phospholamban molecules studied decreased the apparent Ca2+ affinity of the Ca2+ pump, but had no effect on enzyme velocity measured at saturating Ca2+ concentration. Monomeric phospholamban produced by mutations in the leucine/isoleucine zipper domain decreased the apparent Ca2+ affinity the most, giving stronger inhibition of the Ca2+ pump than even wild-type phospholamban. Thus, the baculovirus cell expression system is ideally suited for examining functional interactions between phospholamban and SERCA2a. The results obtained suggest that the phospholamban monomer may be the active species inhibiting the Ca2+ pump in the cardiac sarcoplasmic reticulum membrane.
Autry
J M
JM
Department of Medicine, Indiana University School of Medicine, Indianapolis 46202, USA.
Jones
L R
LR
D000818:Animals; D016367:Baculoviridae; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D004285:Dogs; D007700:Kinetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D018411:Spodoptera; D014162:Transfection; D000099211:Phospholamban
D002118:Calcium; D002135:Calcium-Binding Proteins; D000252:Calcium-Transporting ATPases; D011994:Recombinant Proteins; D000099211:Phospholamban