Department of Pharmacology, University of California, Davis, California, USA.
Biophys J. 2011 Nov 2;101(9):2170-7. doi: 10.1016/j.bpj.2011.09.030. Epub 2011 Nov 1.
Calmodulin (CaM) binding to the type 2 ryanodine receptor (RyR2) regulates Ca release from the cardiac sarcoplasmic reticulum (SR). However, the structural basis of CaM regulation of the RyR2 is poorly defined, and the presence of other potential CaM binding partners in cardiac myocytes complicates resolution of CaM's regulatory interactions with RyR2. Here, we show that a fluorescence-resonance-energy-transfer (FRET)-based approach can effectively resolve RyR2 CaM binding, both in isolated SR membrane vesicles and in permeabilized ventricular myocytes. A small FRET donor was targeted to the RyR2 cytoplasmic assembly via fluorescent labeling of the FKBP12.6 subunit. Acceptor fluorophore was attached at discrete positions within either the N- or the C-lobe of CaM. FRET between FKBP12.6 and CaM bound to SR vesicles indicated CaM binding at a single high-affinity site within 60 Å of FKBP12.6. Micromolar Ca increased the apparent affinity of CaM binding and slowed CaM dissociation, but did not significantly affect maximal FRET efficiency at saturating CaM. FRET was strongest when the acceptor was attached at either of two positions within CaM's N-lobe versus sites in CaM's C-lobe, providing CaM orientation information. In permeabilized ventricular myocytes, FKBP12.6 and CaM colocalized to Z-lines, and the efficiency of energy transfer to both the N- and C-lobes of CaM was comparable to that observed in SR vesicle experiments. Results also indicate that both the location and orientation of CaM binding on the RyR2 are very similar to the skeletal muscle RyR1 isoform. Specific binding of CaM to functional RyR2 channels in the cardiac myocyte environment can be monitored using FKBP biosensors and FRET.
钙调蛋白 (CaM) 与 2 型兰尼碱受体 (RyR2) 结合，调节心肌肌浆网 (SR) 中的 Ca 释放。然而，CaM 对 RyR2 的调节的结构基础还没有明确的定义，而且心脏肌细胞中存在其他潜在的 CaM 结合伴侣，这使得解析 CaM 与 RyR2 的调节相互作用变得复杂。在这里，我们展示了一种基于荧光共振能量转移 (FRET) 的方法可以有效地解析 RyR2 的 CaM 结合，无论是在分离的 SR 膜囊泡中还是在透化的心室肌细胞中。通过 FKBP12.6 亚基的荧光标记，将一个小的 FRET 供体靶向 RyR2 的细胞质组装。受体荧光团连接在 CaM 的 N 或 C 结构域的离散位置上。FKBP12.6 和结合在 SR 囊泡上的 CaM 之间的 FRET 表明 CaM 在距离 FKBP12.6 约 60 Å 的单个高亲和力位点结合。微摩尔 Ca 增加了 CaM 结合的表观亲和力，并减缓了 CaM 的解离，但在饱和 CaM 时，对最大 FRET 效率没有显著影响。当受体连接在 CaM 的 N 结构域的两个位置之一或 CaM 的 C 结构域的位点时，FRET 最强，提供了 CaM 取向信息。在透化的心室肌细胞中，FKBP12.6 和 CaM 共定位于 Z 线，并且能量转移到 CaM 的 N 和 C 结构域的效率与在 SR 囊泡实验中观察到的相似。结果还表明，CaM 结合到 RyR2 上的位置和取向与骨骼肌 RyR1 同工型非常相似。在心脏肌细胞环境中，可以使用 FKBP 生物传感器和 FRET 监测 CaM 与功能性 RyR2 通道的特异性结合。
