Intermolecular conformational coupling and free energy exchange enhance the catalytic efficiency of cardiac muscle SERCA2a following the relief of phospholamban inhibition
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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DOI：
10.1021/bi048011i
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发表时间：
2005-05-31
期刊：
影响因子：
2.9
通讯作者：
Froehlich, JP
中科院分区：
文献类型：
--
作者：
Mahaney, JE;Albers, RW;Froehlich, JP
β(1) 激动剂对心肌肌浆网 Ca2+-ATP 酶 (SERCA2a) 的激活涉及受磷蛋白 (PLB) 的 cAMP 和 PKA 依赖性磷酸化，从而减轻 PLB 对 SERCA2a 的抑制作用。为了研究 SERCA2a 激活的机制，我们将 High Five 昆虫细胞微粒体中含有（+）和不含（-）PLB 的 SERCA2a 表达的动力学特性与天然骨骼肌和心肌 SR 中 SERCA1 和 SERCA2a 的动力学特性进行了比较。天然 SERCA1 和无 PLB 的表达 SERCA2a 均表现出 ATP 前稳态催化位点去磷酸化的高亲和力 (10-50 μM) 激活、ADP 敏感磷酸酶 (E1P) 的稳态积累以及 EGTA 诱导的磷酸酶 (E2P) 水解的快速阶段。相比之下，天然心脏SR囊泡中的SERCA2a以及用PLB表达SERCA2a缺乏ATP的高亲和力激活和E2P水解的快速阶段，并且表现出低稳态水平的EIP。结果表明，骨骼和心脏 SR 之间 Ca2+ 转运的动力学差异是由于心脏 SR 中存在受磷蛋白，而不是由于 SERCA1 和 SERCA2a 之间的异构体依赖性差异。因此，我们根据 PLB 干扰 SERCA2a 寡聚相互作用的模型来讨论结果，这对于骨骼肌 SERCA1 中 Ca2+ 转运的机制很重要 [Mahaney, J. E., Thomas, D. D., and Froehlich, J. P. (2004) Biochemistry 43, 4400-4416]。我们认为催化循环过程中 SERCA2a 分子的分子间偶联对于 Ca2+ 转运活性的变化是必然的，伴随着 PLB 对心脏 SR Ca2+-ATPase 抑制的缓解。
Activation of cardiac muscle sarcoplasmic reticulum Ca2+-ATPase (SERCA2a) by beta(1)-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 mu M) 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 EIP. 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.