Ca2+ binding to site I of the cardiac Ca2+ pump is sufficient to dissociate phospholamban.
Phospholamban (PLB) inhibits the activity of SERCA2a, the Ca(2+)-ATPase in cardiac sarcoplasmic reticulum, by decreasing the apparent affinity of the enzyme for Ca(2+). Recent cross-linking studies have suggested that PLB binding and Ca(2+) binding to SERCA2a are mutually exclusive. PLB binds to the E2 conformation of the Ca(2+)-ATPase, preventing formation of E1, the conformation that binds two Ca(2+) (at sites I and II) with high affinity and is required for ATP hydrolysis. Here we determined whether Ca(2+) binding to site I, site II, or both sites is sufficient to dissociate PLB from the Ca(2+) pump. Seven SERCA2a mutants with amino acid substitutions at Ca(2+)-binding site I (E770Q, T798A, and E907Q), site II (E309Q and N795A), or both sites (D799N and E309Q/E770Q) were made, and the effects of Ca(2+) on N30C-PLB cross-linking to Lys(328) of SERCA2a were measured. In agreement with earlier reports with the skeletal muscle Ca(2+)-ATPase, none of the SERCA2a mutants (except E907Q) hydrolyzed ATP in the presence of Ca(2+); however, all were phosphorylatable by P(i) to form E2P. Ca(2+) inhibition of E2P formation was observed only in SERCA2a mutants retaining site I. In cross-linking assays, strong cross-linking between N30C-PLB and each Ca(2+)-ATPase mutant was observed in the absence of Ca(2+). Importantly, however, micromolar Ca(2+) inhibited PLB cross-linking only to mutants retaining a functional Ca(2+)-binding site I. The dynamic equilibrium between Ca(2+) pumps and N30C-PLB was retained by all mutants, demonstrating normal regulation of cross-linking by ATP, thapsigargin, and anti-PLB antibody. From these results we conclude that site I is the key Ca(2+)-binding site regulating the physical association between PLB and SERCA2a.
Chen
Zhenhui
Z
Department of Medicine, Indiana University School of Medicine, Krannert Institute of Cardiology, Indianapolis, Indiana 46202, USA.
Akin
Brandy L
BL
Jones
Larry R
LR
D000255:Adenosine Triphosphate / Q000737:chemistry; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000737:chemistry*; D002135:Calcium-Binding Proteins / Q000737:chemistry*; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004285:Dogs; D006868:Hydrolysis; D007313:Insecta; D007700:Kinetics; D008956:Models, Chemical; D009154:Mutation; D010766:Phosphorylation; D011485:Protein Binding; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000378:metabolism; D000099211:Phospholamban
D000255:Adenosine Triphosphate; D002118:Calcium; D002135:Calcium-Binding Proteins; D003432:Cross-Linking Reagents; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D000099211:Phospholamban