Zhang Jing, Liu Zheng, Masumiya Haruko, Wang Ruiwu, Jiang Dawei, Li Fei, Wagenknecht Terence, Chen S R Wayne
Cardiovascular Research Group, Department of Physiology, University of Calgary, Alberta T2N 4N1, Canada.
J Biol Chem. 2003 Apr 18;278(16):14211-8. doi: 10.1074/jbc.M213164200. Epub 2003 Feb 7.
Of the three divergent regions of ryanodine receptors (RyRs), divergent region 3 (DR3) is the best studied and is believed to be involved in excitation-contraction coupling as well as in channel regulation by Ca(2+) and Mg(2+). To gain insight into the structural basis of DR3 function, we have determined the location of DR3 in the three-dimensional structure of RyR2. We inserted green fluorescent protein (GFP) into the middle of the DR3 region after Thr-1874 in the sequence. HEK293 cells expressing this GFP-RyR2 fusion protein, RyR2(T1874-GFP,) were readily detected by their green fluorescence, indicating proper folding of the inserted GFP. RyR2(T1874-GFP) was further characterized functionally by assays of Ca(2+) release and [(3)H]ryanodine binding. These analyses revealed that RyR2(T1874-GFP) functions as a caffeine- and ryanodine-sensitive Ca(2+) release channel and displays Ca(2+) dependence and [(3)H]ryanodine binding properties similar to those of the wild type RyR2. RyR2(T1874-GFP) was purified from cell lysates in a single step by affinity chromatography using GST-FKBP12.6 as the affinity ligand. The three-dimensional structure of the purified RyR2(T1874-GFP) was then reconstructed using cryoelectron microscopy and single particle image analysis. Comparison of the three-dimensional reconstructions of wild type RyR2 and RyR2(T1874-GFP) revealed the location of the inserted GFP, and hence the DR3 region, in one of the characteristic domains of RyR, domain 9, in the clamp-shaped structure adjacent to the FKBP12 and FKBP12.6 binding sites. COOH-terminal truncation analysis demonstrated that a region between 1815 and 1855 near DR3 is essential for GST-FKBP12.6 binding. These results provide a structural basis for the role of the DR3 region in excitation-contraction coupling and in channel regulation.
在兰尼碱受体（RyRs）的三个不同区域中，不同区域3（DR3）是研究得最深入的，并且被认为参与兴奋 - 收缩偶联以及Ca(2+)和Mg(2+)对通道的调节。为了深入了解DR3功能的结构基础，我们确定了DR3在RyR2三维结构中的位置。我们将绿色荧光蛋白（GFP）插入到序列中苏氨酸 - 1874之后的DR3区域中间。表达这种GFP - RyR2融合蛋白（RyR2(T1874 - GFP)）的HEK293细胞通过其绿色荧光很容易被检测到，这表明插入的GFP折叠正确。通过Ca(2+)释放和[(3)H]兰尼碱结合测定对RyR2(T1874 - GFP)进行了进一步的功能表征。这些分析表明，RyR2(T1874 - GFP)作为对咖啡因和兰尼碱敏感的Ca(2+)释放通道发挥作用，并且显示出与野生型RyR2相似的Ca(2+)依赖性和[(3)H]兰尼碱结合特性。使用GST - FKBP12.6作为亲和配体，通过亲和色谱从细胞裂解物中一步纯化出RyR2(T1874 - GFP)。然后使用冷冻电子显微镜和单颗粒图像分析重建纯化的RyR2(T1874 - GFP)的三维结构。野生型RyR2和RyR2(T1874 - GFP)的三维重建比较揭示了插入的GFP的位置，从而也揭示了DR3区域在RyR的一个特征结构域，即结构域9中，位于与FKBP12和FKBP12.6结合位点相邻的钳形结构中。COOH末端截短分析表明，DR3附近1815至1855之间的区域对于GST - FKBP12.6结合至关重要。这些结果为DR3区域在兴奋 - 收缩偶联和通道调节中的作用提供了结构基础。
