Ryanodine receptor as a novel therapeutic target of lethal arrhythmia

我们先前证明K201（JTV 5I 9）通过纠正衰竭心脏中兰尼碱受体（RyR 2）的N-末端（0-600）和中心区域（2000-2500）之间的缺陷性结构域间相互作用来抑制Call泄漏。在这里，我们确定了K201结合结构域，并表征了这种新结构域对RyR 2通道门控的作用。使用石英晶体微量天平技术的测定揭示K201特异性结合重组RyR 2片段：1741-2270，但不结合来自1-2750区域的其它RyR 2片段（~ 500个氨基酸残基）。通过进一步分析该片段<1741-2270>，发现K201特异性结合其亚片段<2114-2149>。使用与该亚片段匹配的肽（DP 1<2114-2149>）作为载体，用甲基香豆素乙酸酯（MCA）特异性标记RyR 2。此外，在几个重组RyR 2片段（1-2750）中，仅片段^<2234-2750>被特异性地MCA标记;这表明K201结合结构域^<2114-2149>与结构域^结合<2234-2750>。向<2114-2149>MCA标记的SR中加入DP β干扰了结构域β和结构域β之间的相互作用<2114-2149>，<2234-2750>导致结构域解链，如结合的MCA对大尺寸荧光猝灭剂的可接近性增加所证明的。衰竭心肌细胞（F;与正常（N; n = 15）心肌细胞相比，自发性Ca^&lt;2+&gt;火花频率（CaSF：s-1.100μm-1）显著增加（F：2.7±0.7，N：0.9±0.4; p&lt;0.01 vs N），而DP^掺入<2114-2149>显著降低CaSF（1.3±0.5，p&lt;0.01 vs F），与K201产生的效果相同。总之，我们首先将K201结合位点鉴定为<2114-2149>RyR 2的结构域。结构域和中央结构域之间的结构域间相互作用的中断<2114-2149><2234-2750>似乎介导RyR 2在衰竭心脏中的稳定，这可能导致针对心力衰竭和可能致命的心律失常的新的治疗策略。

We previously demonstrated that K201 (JTV5I9) inhibits the Call leak by correcting the defective inter-domain interaction between N-terminal (0-600) and central regions (2000-2500) of the ryanodine receptor (RyR2) in failing hearts. Here, we identified the K201-binding domain and characterized the role of this novel domain on RyR2 channel gating. An assay using a quartz-crystal microbalance technique revealed that K201 specifically bound to recombinant RyR2 fragment: 1741-2270, but not to other RyR2 fragments (-500 amino acid residues) from the 1-2750 region. By further analysis of the fragment^<1741-2270>, K201 was found to specifically bind to its sub-fragment^<2114-2149>. Using the peptide matching this sub-fragment (DP^<2114-2149>) as a carrier, the RyR2 was specifically labeled with methylcoumarin acetate (MCA). Moreover, of several recombinant RyR2 fragments (1-2750), only fragment^<2234-2750> was specifically MCA-labeled; this suggests that the K201 binding domain^<2114-2149> binds with domain^<2234-2750>. Addition of DP^<2114-2149> to the MCA-labeled SR 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 (F; n=15), the frequency of spontaneous Ca^<2+> spark (CaSF: s-1.100μm-1) was markedly increased compared with normal (N; n=15) cardiomyocytes (F: 2.7±0.7, N: 0.9±0.4; p<0.01 vs N), whereas incorporation of DP^<2114-2149> markedly decreased CaSF (1.3±0.5, p<0.01 vs F), the same effect as that produced by K201. In conclusion, we first identified the K201-binding site as domain^<2114-2149> of RyR2. Interruption of the inter-domain 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.