Intermolecular Conformational Coupling and Free Energy Exchange Enhance the Catalytic Efficiency of Cardiac Muscle SERCA2a following the Relief of Phospholamban Inhibition
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- 作者：James E. Mahaney ; R. Wayne Albers ; Jason R. Waggoner ; Howard C. Kutchai ; and Jeffrey P. Froehlich
- 刊名：Biochemistry
- 出版年：2005
- 出版时间：May 31, 2005
- 年：2005
- 卷：44
- 期：21
- 页码：7713 - 7724
- 全文大小：223K
- 年卷期：v.44,no.21(May 31, 2005)
- ISSN：1520-4995
文摘
Activation of cardiac muscle sarcoplasmic reticulum Ca2+-ATPase (SERCA2a) by 
1-agonistsinvolves cAMP- and PKA-dependent phosphorylation of phospholamban (PLB), which relieves theinhibitory effects of PLB on SERCA2a. To investigate the mechanism of SERCA2a activation, we comparedthe kinetic properties of SERCA2a expressed with (+) and without (-) PLB in High Five insect cellmicrosomes to those of SERCA1 and SERCA2a in native skeletal and cardiac muscle SR. Both nativeSERCA1 and expressed SERCA2a without PLB exhibited high-affinity (10-50 
M) activation of pre-steady-state catalytic site dephosphorylation by ATP, steady-state accumulation of the ADP-sensitivephosphoenzyme (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-affinityactivation 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 dueto the presence of phospholamban in cardiac SR, and not due to isoform-dependent differences betweenSERCA1 and SERCA2a. Therefore, the results are discussed in terms of a model in which PLB interfereswith SERCA2a oligomeric interactions, which are important for the mechanism of Ca2+ transport in skeletalmuscle 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 isobligatory for the changes in Ca2+ transport activity that accompany the relief of PLB inhibition of thecardiac SR Ca2+-ATPase.
1-agonistsinvolves cAMP- and PKA-dependent phosphorylation of phospholamban (PLB), which relieves theinhibitory effects of PLB on SERCA2a. To investigate the mechanism of SERCA2a activation, we comparedthe kinetic properties of SERCA2a expressed with (+) and without (-) PLB in High Five insect cellmicrosomes to those of SERCA1 and SERCA2a in native skeletal and cardiac muscle SR. Both nativeSERCA1 and expressed SERCA2a without PLB exhibited high-affinity (10-50 M) activation of pre-steady-state catalytic site dephosphorylation by ATP, steady-state accumulation of the ADP-sensitivephosphoenzyme (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-affinityactivation 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 dueto the presence of phospholamban in cardiac SR, and not due to isoform-dependent differences betweenSERCA1 and SERCA2a. Therefore, the results are discussed in terms of a model in which PLB interfereswith SERCA2a oligomeric interactions, which are important for the mechanism of Ca2+ transport in skeletalmuscle 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 isobligatory for the changes in Ca2+ transport activity that accompany the relief of PLB inhibition of thecardiac SR Ca2+-ATPase.