Cardiac Calcium ATPase Dimerization Measured by Cross-Linking and Fluorescence Energy Transfer
Cardiac Calcium ATPase Dimerization Measured by Cross-Linking and Fluorescence Energy Transfer
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DOI：
10.1016/j.bpj.2016.08.005
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发表时间：
2016-09-20
影响因子：
3.4
通讯作者：
Robia, Seth L.
中科院分区：
文献类型：
--
作者：
Blackwell, Daniel J.;Zak, Taylor J.;Robia, Seth L.
心肌肌/内质网钙atp酶（SERCA）建立跨越肌浆网膜的细胞内钙梯度。有人提出SERCA形成的同聚物增加了钙运输的催化速率。我们使用光激活交联剂研究了SERCA在兔左心室肌细胞中的二聚化。交联SERCA的Western印迹显示高分子量物种与SERCA寡聚一致。荧光标记SERCA2a瞬时转染细胞的荧光共振能量转移测量显示，SERCA很容易形成同型二聚体。这些二聚体是在SERCA调控伙伴磷蛋白（PLB）缺失或存在的情况下形成的，并且不受PLB磷酸化或钙或ATP变化的影响。荧光寿命数据与PLB与SERCA同型二聚体在1:2的化学计量中相互作用的模型兼容。总之，这些结果表明，SERCA在活细胞中形成组成型二聚体，二聚体的形成不受SERCA构象平衡、PLB结合或PLB磷酸化的调节。
The cardiac sarco/endoplasmic reticulum calcium ATPase (SERCA) establishes the intracellular calcium gradient across the sarcoplasmic reticulum membrane. It has been proposed that SERCA forms homooligomers that increase the catalytic rate of calcium transport. We investigated SERCA dimerization in rabbit left ventricular myocytes using a photoactivatable cross-linker. Western blotting of cross-linked SERCA revealed higher-molecular-weight species consistent with SERCA oligomerization. Fluorescence resonance energy transfer measurements in cells transiently transfected with fluorescently labeled SERCA2a revealed that SERCA readily forms homodimers. These dimers formed in the absence or presence of the SERCA regulatory partner, phospholamban (PLB) and were unaltered by PLB phosphorylation or changes in calcium or ATP. Fluorescence lifetime data are compatible with a model in which PLB interacts with a SERCA homodimer in a stoichiometry of 1:2. Together, these results suggest that SERCA forms constitutive homodimers in live cells and that dimer formation is not modulated by SERCA conformational poise, PLB binding, or PLB phosphorylation.