Residues 2-25 of phospholamban are insufficient to inhibit Ca2+ transport ATPase of cardiac sarcoplasmic reticulum.
We have used sarcoplasmic reticulum (SR) vesicles isolated from AT-1 cardiomyocytes to examine the mechanism of phospholamban (PLB) inhibition of the Ca2+ pump of cardiac SR. By immunoblotting with monoclonal antibodies, we observed that these SR vesicles contained a normal amount of the cardiac isoform of the Ca2+ pump (SERCA 2) but only a trace level of PLB. A monoclonal antibody that recognized amino acid residues 9-17 of PLB had no significant effect on Ca2+ transport by AT-1 SR vesicles, but it increased Ca2+ transport into mouse ventricular SR vesicles greater than 10-fold by reversing PLB inhibition of SERCA 2 at low ionized Ca2+ concentration. To further explore the domains of PLB responsible for SERCA 2 inhibition, we examined the effect of a PLB synthetic peptide consisting of amino acid residues 2-25 on Ca2+ uptake by AT-1 SR vesicles. Even at concentrations as high as 0.44 mM, no significant effect of the peptide was observed. Based on these results, we conclude that the cytoplasmic domain of PLB, containing the phosphorylation sites, by itself is insufficient to inhibit the Ca2+ pump and that the transmembrane region, which stabilizes the pentamer, is also essential for Ca2+ transport regulation.
Jones
L R
LR
Department of Medicine, Indiana University School of Medicine, Indianapolis 46202.
Field
L J
LJ
D000251:Adenosine Triphosphatases / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D001692:Biological Transport; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D002135:Calcium-Binding Proteins / Q000494:pharmacology*; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors*; D006325:Heart Atria; D006352:Heart Ventricles; D007527:Isoenzymes / Q000037:antagonists & inhibitors*; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D009206:Myocardium / Q000201:enzymology*; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000494:pharmacology*; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D013997:Time Factors; D000099211:Phospholamban
D000251:Adenosine Triphosphatases; D000911:Antibodies, Monoclonal; D002118:Calcium; D002135:Calcium-Binding Proteins; D000252:Calcium-Transporting ATPases; D007527:Isoenzymes; D010446:Peptide Fragments; D000099211:Phospholamban