Wadsworth Center, New York State Department of Health, Albany, New York 12201, USA.
J Cell Sci. 2013 Oct 1;126(Pt 19):4527-35. doi: 10.1242/jcs.133454. Epub 2013 Jul 18.
Calmodulin (CaM), a 16 kDa ubiquitous calcium-sensing protein, is known to bind tightly to the calcium release channel/ryanodine receptor (RyR), and modulate RyR function. CaM binding studies using RyR fragments or synthetic peptides have revealed the presence of multiple, potential CaM-binding regions in the primary sequence of RyR. In the present study, we inserted GFP into two of these proposed CaM-binding sequences and mapped them onto the three-dimensional structure of intact cardiac RyR2 by cryo-electron microscopy. Interestingly, we found that the two potential CaM-binding regions encompassing, Arg3595 and Lys4269, respectively, are in close proximity and are adjacent to the previously mapped CaM-binding sites. To monitor the conformational dynamics of these CaM-binding regions, we generated a fluorescence resonance energy transfer (FRET) pair, a dual CFP- and YFP-labeled RyR2 (RyR2R3595-CFP/K4269-YFP) with CFP inserted after Arg3595 and YFP inserted after Lys4269. We transfected HEK293 cells with the RyR2R3595-CFP/K4269-YFP cDNA, and examined their FRET signal in live cells. We detected significant FRET signals in transfected cells that are sensitive to the channel activator caffeine, suggesting that caffeine is able to induce conformational changes in these CaM-binding regions. Importantly, no significant FRET signals were detected in cells co-transfected with cDNAs encoding the single CFP (RyR2R3595-CFP) and single YFP (RyR2K4269-YFP) insertions, indicating that the FRET signal stemmed from the interaction between R3595-CFP and K4269-YFP that are in the same RyR subunit. These observations suggest that multiple regions in the RyR2 sequence may contribute to an intra-subunit CaM-binding pocket that undergoes conformational changes during channel gating.
钙调蛋白（CaM）是一种普遍存在的 16kDa 钙感应蛋白，已知其与钙释放通道/兰尼碱受体（RyR）紧密结合，并调节 RyR 功能。使用 RyR 片段或合成肽进行的 CaM 结合研究表明，RyR 的一级序列中存在多个潜在的 CaM 结合区域。在本研究中，我们将 GFP 插入这两个拟议的 CaM 结合序列中，并通过冷冻电镜将它们映射到完整的心脏 RyR2 的三维结构上。有趣的是，我们发现两个潜在的 CaM 结合区域分别包含 Arg3595 和 Lys4269，它们彼此靠近，并且紧邻先前映射的 CaM 结合位点。为了监测这些 CaM 结合区域的构象动力学，我们生成了一个荧光共振能量转移（FRET）对，一个双重 CFP-和 YFP 标记的 RyR2（RyR2R3595-CFP/K4269-YFP），其中 CFP 插入 Arg3595 之后，YFP 插入 Lys4269 之后。我们用 RyR2R3595-CFP/K4269-YFP cDNA 转染 HEK293 细胞，并在活细胞中检查它们的 FRET 信号。我们在转染细胞中检测到了显著的 FRET 信号，这些信号对通道激活剂咖啡因敏感，表明咖啡因能够诱导这些 CaM 结合区域的构象变化。重要的是，在共转染编码单个 CFP（RyR2R3595-CFP）和单个 YFP（RyR2K4269-YFP）插入物的 cDNA 的细胞中未检测到显著的 FRET 信号，表明 FRET 信号源自同一 RyR 亚基中的 R3595-CFP 和 K4269-YFP 之间的相互作用。这些观察结果表明，RyR2 序列中的多个区域可能有助于一个亚基内的 CaM 结合口袋，该口袋在通道门控过程中发生构象变化。
