Ca2+ binding to site I of the cardiac Ca2+ pump is sufficient to dissociate phospholamban.
ABSTRACT 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.
Article: Spatial and dynamic interactions between phospholamban and the canine cardiac Ca2+ pump revealed with use of heterobifunctional cross-linking agents.[show abstract] [hide abstract]
ABSTRACT: Heterobifunctional thiol to amine cross-linking agents were used to gain new insights on the dynamics and conformational factors governing the interaction between the cardiac Ca2+ pump (SERCA2a) and phospholamban (PLB). PLB is a small protein inhibitor of SERCA2a that reduces enzyme affinity for Ca2+ and thereby regulates cardiac contractility. We found that the PLB monomer with Asn27 or Asn30 changed to Cys (N27C-PLB or N30C-PLB) cross-linked to lysine of SERCA2a within seconds with > or =80% efficiency. Optimal cross-linking occurred at spacer chain lengths of 10 and 15 A for N27C and N30C, respectively. The rapid time course of cross-linking indicated that neither dissociation of PLB pentamers nor binding of PLB monomers to SERCA2a was rate-limiting. Cross-linking occurred only to the E2 (Ca2+-free) conformation of SERCA2a, was strongly favored by nucleotide binding to this state, and was completely inhibited by thapsigargin. Protein sequencing in combination with mutagenesis identified of Lys328 of SERCA2a as the target of cross-linking. A three-dimensional map of interacting residues indicated that the cross-linking distances were entirely compatible with the 10-A distance recently determined between N30C of PLB and Cys318 of SERCA2a. In contrast, Lys3 of PLB did not cross-link to any Lys (or Cys) of SERCA2a, suggesting that previous three-dimensional models that constrain Lys3 near residues 397-400 of thapsigargin-inhibited SERCA2a should be viewed with caution. Furthermore, although earlier models of PLB.SERCA2a are based on thapsigargin-bound SERCA, our results suggest that the nucleotide-bound, E2 conformation is substantially different and represents the key conformational state for interacting with PLB.Journal of Biological Chemistry 12/2003; 278(48):48348-56. · 4.77 Impact Factor
Article: Functional Co-expression of the canine cardiac Ca2+ pump and phospholamban in Spodoptera frugiperda (Sf21) cells reveals new insights on ATPase regulation.[show abstract] [hide abstract]
ABSTRACT: The utility of the baculovirus cell expression system for investigating Ca2+-ATPase and phospholamban regulatory interactions was examined. cDNA encoding the canine cardiac sarco(endo)plasmic Ca2+-ATPase pump (SERCA2a) was cloned for the first time and expressed in the presence and absence of phospholamban in Spodoptera frugiperda (Sf21) insect cells. The recombinant Ca2+ pump was produced in high yield, contributing 20% of the total membrane protein in Sf21 microsomes. At least 70% of the expressed pumps were active. Co-expression of wild-type, pentameric phospholamban with the Ca2+-ATPase decreased the apparent affinity of the ATPase for Ca2+, but had no effect on the maximum velocity of the enzyme, similar to phospholamban's action in cardiac sarcoplasmic reticulum vesicles. To investigate the importance of the oligomeric structure of phospholamban in ATPase regulation, SERCA2a was co-expressed with a monomeric mutant of phospholamban, in which leucine residue 37 was changed to alanine. Surprisingly, monomeric phospholamban suppressed SERCA2a Ca2+ affinity more strongly than did wild-type phospholamban, demonstrating that the pentamer is not essential for Ca2+ pump inhibition and that the monomer is the more active species. To test if phospholamban functions as a Ca2+ channel, Sf21 microsomes expressing either SERCA2a or SERCA2a plus phospholamban were actively loaded with Ca2+ and then assayed for unidirectional 45Ca2+ efflux. No evidence for a Ca2+ channel activity of phospholamban was obtained. We conclude that the phospholamban monomer is an important regulatory component inhibiting SERCA2a in cardiac sarcoplasmic reticulum membranes, and that the channel activity of phospholamban previously observed in planar bilayers is not involved in the mechanism of ATPase regulation.Journal of Biological Chemistry 07/1997; 272(25):15872-80. · 4.77 Impact Factor
Article: Mechanism of reversal of phospholamban inhibition of the cardiac Ca2+-ATPase by protein kinase A and by anti-phospholamban monoclonal antibody 2D12.[show abstract] [hide abstract]
ABSTRACT: Our model of phospholamban (PLB) regulation of the cardiac Ca(2+)-ATPase in sarcoplasmic reticulum (SERCA2a) states that PLB binds to the Ca(2+)-free, E2 conformation of SERCA2a and blocks it from transitioning from E2 to E1, the Ca(2+)-bound state. PLB and Ca(2+) binding to SERCA2a are mutually exclusive, and PLB inhibition of SERCA2a is manifested as a decreased apparent affinity of SERCA2a for Ca(2+). Here we extend this model to explain the reversal of SERCA2a inhibition that occurs after phosphorylation of PLB at Ser(16) by protein kinase A (PKA) and after binding of the anti-PLB monoclonal antibody 2D12, which recognizes residues 7-13 of PLB. Site-specific cysteine variants of PLB were co-expressed with SERCA2a, and the effects of PKA phosphorylation and 2D12 on Ca(2+)-ATPase activity and cross-linking to SERCA2a were monitored. In Ca(2+)-ATPase assays, PKA phosphorylation and 2D12 partially and completely reversed SERCA2a inhibition by decreasing K(Ca) values for enzyme activation, respectively. In cross-linking assays, cross-linking of PKA-phosphorylated PLB to SERCA2a was inhibited at only two of eight sites when conducted in the absence of Ca(2+) favoring E2. However, at a subsaturating Ca(2+) concentration supporting some E1, cross-linking of phosphorylated PLB to SERCA2a was attenuated at all eight sites. K(Ca) values for cross-linking inhibition were decreased nearly 2-fold at all sites by PLB phosphorylation, demonstrating that phosphorylated PLB binds more weakly to SERCA2a than dephosphorylated PLB. In parallel assays, 2D12 blocked PLB cross-linking to SERCA2a at all eight sites regardless of Ca(2+) concentration. Our results demonstrate that 2D12 restores maximal Ca(2+)-ATPase activity by physically disrupting the binding interaction between PLB and SERCA2a. Phosphorylation of PLB by PKA weakens the binding interaction between PLB and SERCA2a (yielding more PLB-free SERCA2a molecules at intermediate Ca(2+) concentrations), only partially restoring Ca(2+) affinity and Ca(2+)-ATPase activity.Journal of Biological Chemistry 08/2007; 282(29):20968-76. · 4.77 Impact Factor