From the Department of Chemistry "Ugo Schiff," University of Florence, 50019 Sesto Fiorentino, Italy and.
Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.
J Biol Chem. 2017 Dec 29;292(52):21330-21339. doi: 10.1074/jbc.M117.794453. Epub 2017 Oct 29.
The sarcoplasmic reticulum Ca-ATPase SERCA promotes muscle relaxation by pumping calcium ions from the cytoplasm into the sarcoplasmic reticulum. SERCA activity is regulated by a variety of small transmembrane peptides, most notably by phospholamban in cardiac muscle and sarcolipin in skeletal muscle. However, how phospholamban and sarcolipin regulate SERCA is not fully understood. In the present study, we evaluated the effects of phospholamban and sarcolipin on calcium translocation and ATP hydrolysis by SERCA under conditions that mimic environments in sarcoplasmic reticulum membranes. For pre-steady-state current measurements, proteoliposomes containing SERCA and phospholamban or sarcolipin were adsorbed to a solid-supported membrane and activated by substrate concentration jumps. We observed that phospholamban altered ATP-dependent calcium translocation by SERCA within the first transport cycle, whereas sarcolipin did not. Using pre-steady-state charge (calcium) translocation and steady-state ATPase activity under substrate conditions (various calcium and/or ATP concentrations) promoting particular conformational states of SERCA, we found that the effect of phospholamban on SERCA depends on substrate preincubation conditions. Our results also indicated that phospholamban can establish an inhibitory interaction with multiple SERCA conformational states with distinct effects on SERCA's kinetic properties. Moreover, we noted multiple modes of interaction between SERCA and phospholamban and observed that once a particular mode of association is engaged it persists throughout the SERCA transport cycle and multiple turnover events. These observations are consistent with conformational memory in the interaction between SERCA and phospholamban, thus providing insights into the physiological role of phospholamban and its regulatory effect on SERCA transport activity.
肌浆网 Ca2+-ATP 酶 SERCA 通过将细胞质中的钙离子泵入肌浆网来促进肌肉松弛。SERCA 的活性受多种小跨膜肽的调节，其中最显著的是心肌中的肌浆球蛋白和骨骼肌中的肌浆球蛋白。然而，肌浆球蛋白和肌浆球蛋白如何调节 SERCA 还不完全清楚。在本研究中，我们评估了肌浆球蛋白和肌浆球蛋白在模拟肌浆网膜环境的条件下对 SERCA 钙转运和 ATP 水解的影响。对于预稳定态电流测量，含有 SERCA 和肌浆球蛋白或肌浆球蛋白的蛋白脂质体被吸附到固体支持膜上，并通过底物浓度跳跃激活。我们观察到肌浆球蛋白在第一个转运循环中改变了 SERCA 依赖于 ATP 的钙转运，而肌浆球蛋白没有。使用预稳定态电荷（钙）转运和稳态 ATPase 活性在促进 SERCA 特定构象状态的底物条件下（各种钙和/或 ATP 浓度），我们发现肌浆球蛋白对 SERCA 的影响取决于底物预孵育条件。我们的结果还表明，肌浆球蛋白可以与多个具有不同影响 SERCA 动力学特性的 SERCA 构象状态建立抑制性相互作用。此外，我们注意到 SERCA 和肌浆球蛋白之间存在多种相互作用模式，并观察到一旦特定的缔合模式被参与，它将贯穿整个 SERCA 转运循环和多个周转事件。这些观察结果与 SERCA 和肌浆球蛋白之间的构象记忆一致，从而为肌浆球蛋白的生理作用及其对 SERCA 转运活性的调节作用提供了深入的了解。
