Libin Cardiovascular Institute of Alberta, Department of Physiology and Pharmacology and Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta T2N 4N1, Canada.
J Biol Chem. 2013 Feb 8;288(6):4066-75. doi: 10.1074/jbc.M112.427864. Epub 2012 Dec 20.
Global conformational changes in the three-dimensional structure of the Ca(2+) release channel/ryanodine receptor (RyR) occur upon ligand activation. A number of ligands are able to activate the RyR channel, but whether these structurally diverse ligands induce the same or different conformational changes in the channel is largely unknown. Here we constructed a fluorescence resonance energy transfer (FRET)-based probe by inserting a CFP after residue Ser-2367 and a YFP after residue Tyr-2801 in the cardiac RyR (RyR2) to yield a CFP- and YFP-dual labeled RyR2 (RyR2(Ser-2367-CFP/Tyr-2801-YFP)). Both of these insertion sites have previously been mapped to the "clamp" region in the four corners of the square-shaped cytoplasmic assembly of the three-dimensional structure of RyR2. Using this novel FRET probe, we monitored the extent of conformational changes in the clamp region of RyR2(Ser-2367-CFP/Tyr-2801-YFP) induced by various ligands. We also monitored the extent of Ca(2+) release induced by the same ligands in HEK293 cells expressing RyR2(Ser-2367-CFP/Tyr-2801-YFP). We detected conformational changes in the clamp region for the ligands caffeine, aminophylline, theophylline, ATP, and ryanodine but not for Ca(2+) or 4-chloro-m-cresol, although they all induced Ca(2+) release. Interestingly, caffeine is able to induce further conformational changes in the clamp region of the ryanodine-modified channel, suggesting that ryanodine does not lock RyR in a fixed conformation. Our data demonstrate that conformational changes in the clamp region of RyR are ligand-dependent and suggest the existence of multiple ligand dependent RyR activation mechanisms associated with distinct conformational changes.
钙释放通道/兰尼碱受体（RyR）的三维结构的整体构象变化发生在配体激活时。许多配体能够激活 RyR 通道，但这些结构多样的配体是否在通道中诱导相同或不同的构象变化在很大程度上尚不清楚。在这里，我们通过在心脏 RyR（RyR2）的残基 Ser-2367 后插入 CFP 和 Tyr-2801 后插入 YFP，构建了一种荧光共振能量转移（FRET）探针，从而产生了 CFP 和 YFP 双标记 RyR2（RyR2（Ser-2367-CFP/Tyr-2801-YFP））。这两个插入位点以前都映射到 RyR2 三维结构的四个角的“夹”区域的细胞质组装中。使用这种新型 FRET 探针，我们监测了各种配体诱导的 RyR2（Ser-2367-CFP/Tyr-2801-YFP）的夹区构象变化的程度。我们还监测了相同配体在表达 RyR2（Ser-2367-CFP/Tyr-2801-YFP）的 HEK293 细胞中诱导的 Ca2+释放的程度。我们检测到夹区的构象变化咖啡因，氨茶碱，茶碱，ATP 和兰尼碱的配体，但不是 Ca2+或 4-氯-间甲酚，尽管它们都诱导 Ca2+释放。有趣的是，咖啡因能够诱导兰尼碱修饰的通道夹区的进一步构象变化，这表明兰尼碱不会将 RyR 锁定在固定构象中。我们的数据表明 RyR 夹区的构象变化是配体依赖性的，并表明存在与不同构象变化相关的多种配体依赖性 RyR 激活机制。
