C.N.R. Institute of Molecular Biology and Pathology, Department of Biochemical Sciences A. Rossi Fanelli, Sapienza University of Rome, 00185 Rome, Italy.
J Mol Cell Cardiol. 2010 Jul;49(1):132-41. doi: 10.1016/j.yjmcc.2010.03.003. Epub 2010 Mar 15.
Sorcin is a penta-EF-hand protein that interacts with intracellular target proteins after Ca(2+) binding. The sarcolemmal Na(+)/Ca(2+) exchanger (NCX1) may be an important sorcin target in cardiac muscle. In this study, RNAi knockdown of sorcin, purified sorcin or sorcin variants was employed in parallel measurements of: (i) NCX activity in isolated rabbit cardiomyocytes using electrophysiological techniques and (ii) sorcin binding to the NCX1 calcium binding domains (CBD1 and (iii) using surface plasmon resonance and gel overlay techniques. Sorcin is activated by Ca(2+) binding to the EF3 and EF2 regions, which are connected by the D helix. To investigate the importance of this region in the interaction with NCX1, three variants were examined: W105G and W99G, mutated respectively near EF3 and EF2, and E124A that does not bind Ca(2+) due to a mutation at EF3. Downregulation of sorcin decreased and supplementation with wt sorcin (3muM) increased NCX activity in isolated cardiomyocytes. The relative stimulatory effects of the sorcin variants were: W105G>wt sorcin>Sorcin Calcium Binding Domain (SCBD)>W99G>E124A. Sorcin binding to both CBD1 and 2 was observed. In the presence of 50microM Ca(2+), the interaction with CBD1 followed the order W105G>SCBD>wt sorcin>W99G>E124A. In sorcin, the interacting surface can be mapped on the C-terminal Ca(2+)-binding domain in the D helix region comprising W99. The fast association/dissociation rates that characterize the interaction of sorcin with CBD1 and 2 may permit complex formation/dissociation during an excitation/contraction cycle.
索辛是一种五 EF 手蛋白，在与钙结合后与细胞内靶蛋白相互作用。肌质网钠钙交换体（NCX1）可能是心肌中重要的索辛靶标。在这项研究中，使用 RNAi 敲低、纯化的索辛或索辛变体平行测量：（i）使用电生理技术在分离的兔心肌细胞中测量 NCX 活性，（ii）使用表面等离子体共振和凝胶覆盖技术测量索辛与 NCX1 钙结合域（CBD1）的结合，（iii）使用表面等离子体共振和凝胶覆盖技术测量索辛与 NCX1 钙结合域（CBD1）的结合。索辛通过与 EF3 和 EF2 区域结合而被 Ca2+激活，EF3 和 EF2 区域通过 D 螺旋连接。为了研究该区域在与 NCX1 相互作用中的重要性，研究了三种变体：W105G 和 W99G 分别在 EF3 和 EF2 附近突变，E124A 由于 EF3 中的突变而不结合 Ca2+。索辛的下调减少，而用 wt 索辛（3μM）补充则增加了分离的心肌细胞中的 NCX 活性。索辛变体的相对刺激作用为：W105G>wt 索辛>Sorcin 钙结合域（SCBD）>W99G>E124A。观察到索辛与 CBD1 和 2 均结合。在 50μM Ca2+存在下，与 CBD1 的相互作用顺序为 W105G>SCBD>wt 索辛>W99G>E124A。在索辛中，相互作用的表面可以映射到 D 螺旋区域的 C 端钙结合域上，该区域包含 W99。索辛与 CBD1 和 2 的相互作用具有快速的结合/解离速率，这可能允许在兴奋/收缩循环期间形成/解离复合物。
