Ca2+ Binding to Site I of the Cardiac Ca2+ Pump Is Sufficient to Dissociate Phospholamban
Ca2+ Binding to Site I of the Cardiac Ca2+ Pump Is Sufficient to Dissociate Phospholamban
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DOI：
10.1074/jbc.m109.080820
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发表时间：
2010-01-29
影响因子：
4.8
通讯作者：
Jones, Larry R.
中科院分区：
文献类型：
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作者：
Chen, Zhenhui;Akin, Brandy L.;Jones, Larry R.
磷蛋白（PLB）抑制心脏肌浆网Ca2+- atp酶SERCA2a的活性，通过降低酶对Ca2+的表观亲和力。最近的交联研究表明，PLB结合和Ca2+结合SERCA2a是相互排斥的。PLB结合Ca2+-ATP酶的E2构象，阻止E1的形成，E1是高亲和力结合两个Ca2+（在位点I和II）的构象，是ATP水解所必需的。在这里，我们确定Ca2+结合位点I、位点II或两个位点是否足以将PLB与Ca2+泵分离。制备了7个Ca2+结合位点I （E770Q、T798A和E907Q）、位点II （E309Q和N795A）或两个位点（D799N和E309Q/E770Q）发生氨基酸置换的SERCA2a突变体，并测定了Ca2+对N30C-PLB与SERCA2a Lys328交联的影响。与之前关于骨骼肌Ca2+-ATP酶的报道一致，没有SERCA2a突变体（E907Q除外）在Ca2+存在下水解ATP；然而，它们都能被π磷酸化形成E2P。Ca2+对E2P形成的抑制作用仅在SERCA2a突变体保留位点i中观察到。在交联实验中，在没有Ca2+的情况下，观察到N30C-PLB与每个Ca2+- atp酶突变体之间存在强交联。然而，重要的是，微摩尔Ca2+仅抑制PLB交联到保留功能Ca2+结合位点i的突变体。所有突变体都保留了Ca2+泵和N30C-PLB之间的动态平衡，表明ATP、thapsigargin和抗PLB抗体对交联的正常调节。从这些结果我们得出结论，位点I是调节PLB和SERCA2a之间物理关联的关键Ca2+结合位点。
Phospholamban (PLB) inhibits the activity of SERCA2a, the Ca2+-ATPase in cardiac sarcoplasmic reticulum, by decreasing the apparent affinity of the enzyme for Ca2+. Recent cross-linking studies have suggested that PLB binding and Ca2+ binding to SERCA2a are mutually exclusive. PLB binds to the E2 conformation of the Ca2+-ATPase, preventing formation of E1, the conformation that binds two Ca2+ (at sites I and II) with high affinity and is required for ATP hydrolysis. Here we determined whether Ca2+ binding to site I, site II, or both sites is sufficient to dissociate PLB from the Ca2+ pump. Seven SERCA2a mutants with amino acid substitutions at Ca2+-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 Ca2+ on N30C-PLB cross-linking to Lys328 of SERCA2a were measured. In agreement with earlier reports with the skeletal muscle Ca2+-ATPase, none of the SERCA2a mutants (except E907Q) hydrolyzed ATP in the presence of Ca2+; however, all were phosphorylatable by Pi to form E2P. Ca2+ 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 Ca2+-ATPase mutant was observed in the absence of Ca2+. Importantly, however, micromolar Ca2+ inhibited PLB cross-linking only to mutants retaining a functional Ca2+-binding site I. The dynamic equilibrium between Ca2+ 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 Ca2+-binding site regulating the physical association between PLB and SERCA2a.