Identification of target domains of the cardiac ryanodine receptor to correct channel disorder in failing hearts
Identification of target domains of the cardiac ryanodine receptor to correct channel disorder in failing hearts
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DOI：
10.1161/circulationaha.107.718957
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发表时间：
2008-02-12
期刊：
影响因子：
37.8
通讯作者：
Matsuzaki, Masunori
中科院分区：
文献类型：
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作者：
Yamamoto, Takeshi;Yano, Masafumi;Matsuzaki, Masunori
我们之前已经证明，ryanodine受体2 （RyR2）的n端（0至600）和中心区域（2000至2500）之间有缺陷的区域间相互作用诱导衰竭心脏中的Ca2+泄漏，K201 （JTV519）通过纠正有缺陷的区域间相互作用来抑制Ca2+泄漏。在本报告中，我们确定了k201结合结构域，并表征了该新结构域在RyR2通道调控中的作用。方法和结果：采用石英晶体微天平技术（一种非常灵敏的质量测量技术）测定发现，K201特异性结合重组RyR2片段1741 ~ 2270和1981 ~ 2520，而不与1 ~ 2750区（1 ~ 610、494 ~ 1000、741 ~ 1260、985 ~ 1503、1245 ~ 1768、2234 ~ 2750）的其他RyR2片段结合。通过对该片段（1741-2270）的进一步分析，发现K201特异性地与它的亚片段（2114-2149）结合。利用与该亚片段匹配的肽（DP2114-2149）作为载体，用醋酸甲基香豆素（MCA）以定点方式对RyR2进行荧光标记。经过胰蛋白酶消化后，通过针对中心区域（Ab(2132)）的抗体检测到RyR2的主要mca标记片段（155 kDa）。此外，在多个重组RyR2片段中，只有片段（2234-2750）被特异性MCA标记；这表明k201结合结构域（2114-2149）与结构域（2234-2750）结合。将DP2114-2149添加到MCA标记的肌浆网中会干扰结构域（2114-2149）和结构域（2234-2750）之间的相互作用，导致结构域解压缩，这可以通过结合的MCA对大尺寸荧光猝灭剂的可及性增加来证明。在衰竭心肌细胞中，自发Ca2+火花的频率明显高于正常心肌细胞，而DP2114-2149的掺入显著降低了自发Ca2+火花的频率。结论：我们首先确定了k201结合位点为RyR2的2114-2149结构域。区域（2114-2149）和中心区域（2234-2750）之间区域间相互作用的中断似乎介导了衰竭心脏中RyR2的稳定，这可能导致一种新的治疗心力衰竭和致命性心律失常的策略。
Background-We previously demonstrated that defective interdomain interaction between N-terminal (0 to 600) and central regions (2000 to 2500) of ryanodine receptor 2 (RyR2) induces Ca2+ leak in failing hearts and that K201 (JTV519) inhibits the Ca2+ leak by correcting the defective interdomain interaction. In the present report, we identified the K201-binding domain and characterized the role of this novel domain in the regulation of the RyR2 channel.Methods and Results-An assay using a quartz-crystal microbalance technique (a very sensitive mass-measuring technique) revealed that K201 specifically bound to recombinant RyR2 fragments 1741 to 2270 and 1981 to 2520 but not to other RyR2 fragments from the 1 to 2750 region (1 to 610, 494 to 1000, 741 to 1260, 985 to 1503, 1245 to 1768, 2234 to 2750). By further analysis of the fragment(1741-2270), K201 was found to specifically bind to its subfragment(2114-2149). With the use of the peptide matching this subfragment (DP2114-2149) as a carrier, the RyR2 was fluorescently labeled with methylcoumarin acetate (MCA) in a site-directed manner. After tryptic digestion, the major MCA-labeled fragment of RyR2 (155 kDa) was detected by an antibody raised against the central region (Ab(2132)). Moreover, of several recombinant RyR2 fragments, only fragment(2234-2750) was specifically MCA labeled; this suggests that the K201-binding domain(2114-2149) binds with domain(2234-2750). Addition of DP2114-2149 to the MCA-labeled sarcoplasmic reticulum interfered with the interaction between domain(2114-2149) and domain(2234-2750), causing domain unzipping, as evidenced by an increased accessibility of the bound MCA to a large-size fluorescence quencher. In failing cardiomyocytes, the frequency of spontaneous Ca2+ spark was markedly increased compared with normal cardiomyocytes, whereas incorporation of DP2114-2149 markedly decreased the frequency of spontaneous Ca2+ spark.Conclusions-We first identified the K201-binding site as domain(2114-2149) of RyR2. Interruption of the interdomain interaction between the domain(2114-2149) and central domain(2234-2750) seems to mediate stabilization of RyR2 in failing hearts, which may lead to a novel therapeutic strategy against heart failure and perhaps lethal arrhythmia.