Intermolecular conformational coupling and free energy exchange enhance the catalytic efficiency of cardiac muscle SERCA2a following the relief of phospholamban inhibition.
Activation of cardiac muscle sarcoplasmic reticulum Ca2+-ATPase (SERCA2a) by beta1-agonists involves cAMP- and PKA-dependent phosphorylation of phospholamban (PLB), which relieves the inhibitory effects of PLB on SERCA2a. To investigate the mechanism of SERCA2a activation, we compared the kinetic properties of SERCA2a expressed with (+) and without (-) PLB in High Five insect cell microsomes to those of SERCA1 and SERCA2a in native skeletal and cardiac muscle SR. Both native SERCA1 and expressed SERCA2a without PLB exhibited high-affinity (10-50 microM) activation of pre-steady-state catalytic site dephosphorylation by ATP, steady-state accumulation of the ADP-sensitive phosphoenzyme (E1P), and a rapid phase of EGTA-induced phosphoenzyme (E2P) hydrolysis. In contrast, SERCA2a in native cardiac SR vesicles and expressed SERCA2a with PLB lacked the high-affinity activation by ATP and the rapid phase of E2P hydrolysis, and exhibited low steady-state levels of E1P. The results indicate that the kinetic differences in Ca2+ transport between skeletal and cardiac SR are due to the presence of phospholamban in cardiac SR, and not due to isoform-dependent differences between SERCA1 and SERCA2a. Therefore, the results are discussed in terms of a model in which PLB interferes with SERCA2a oligomeric interactions, which are important for the mechanism of Ca2+ transport in skeletal muscle SERCA1 [Mahaney, J. E., Thomas, D. D., and Froehlich, J. P. (2004) Biochemistry 43, 4400-4416]. We propose that intermolecular coupling of SERCA2a molecules during catalytic cycling is obligatory for the changes in Ca2+ transport activity that accompany the relief of PLB inhibition of the cardiac SR Ca2+-ATPase.
Mahaney
James E
JE
Biomedical Science Division, Edward Via Virginia College of Osteopathic Medicine, Blacksburg, Virginia 24060, USA.
Albers
R Wayne
RW
Waggoner
Jason R
JR
Kutchai
Howard C
HC
Froehlich
Jeffrey P
JP
D000244:Adenosine Diphosphate / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry; D002118:Calcium / Q000378:metabolism; D002132:Calcium Radioisotopes / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000276:immunology; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D002384:Catalysis; D002460:Cell Line; D004285:Dogs; D004533:Egtazic Acid / Q000737:chemistry; D004789:Enzyme Activation; D007425:Intracellular Membranes / Q000201:enzymology; D007527:Isoenzymes / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D009036:Moths; D018482:Muscle, Skeletal / Q000201:enzymology*; D032386:Myoblasts, Cardiac / Q000201:enzymology*; D009206:Myocardium / Q000201:enzymology*; D010766:Phosphorylation; D011487:Protein Conformation; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D013816:Thermodynamics; D000099211:Phospholamban
D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000911:Antibodies, Monoclonal; D002118:Calcium; D002132:Calcium Radioisotopes; D002135:Calcium-Binding Proteins; D000252:Calcium-Transporting ATPases; D004533:Egtazic Acid; D007527:Isoenzymes; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D000099211:Phospholamban