Dantrolene stabilizes domain interactions within the ryanodine receptor
Dantrolene stabilizes domain interactions within the ryanodine receptor
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DOI：
10.1074/jbc.m408375200
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发表时间：
2005-02-25
影响因子：
4.8
通讯作者：
Ikemoto, N
中科院分区：
文献类型：
--
作者：
Kobayashi, S;Bannister, ML;Ikemoto, N
作为“结构域开关”的 N 端和中心结构域之间的结构域间相互作用被认为对于骨骼肌兰尼碱受体 1 Ca2+ 通道的功能调节至关重要。恶性高热（MH）（对挥发性麻醉剂的遗传敏感性）中结构域开关的突变不稳定会导致通道功能不稳定。 Dantrolene 是一种用于治疗 MH 的药物，它与这个拟议的结构域开关内的一个区域结合。为了探索其作用机制，研究了丹曲林对合成结构域肽 DP4 或抗 DP4 抗体激活 MH 样通道的影响。使用 DP4 作为递送载体，将荧光探针甲基香豆素乙酸酯共价连接到结构域开关。结构域解链的程度由甲基香豆素乙酸酯对大分子荧光淬灭剂的可及性确定。 Stern-Volmer 猝灭常数 (K-Q) 随着 DP4 或抗 DP4 抗体的添加而增加。这种增加在 37 和 22 摄氏度下都可以被丹曲林逆转，并且不受钙调蛋白的影响。 [H-3]Ryanodine 与肌浆网的结合和肌浆网 Ca2+ 释放的激活（这两种通道激活的措施）均被 DP4 增强。这些活性在 37°C 时会被丹曲林抑制，但需要在 22°C 下存在钙调蛋白。这些结果表明，丹曲林的作用机制涉及稳定结构域开关内的结构域相互作用，防止结构域解压缩引起的通道功能障碍。我们认为温度和钙调蛋白主要影响结构域开关和 Ca2+ 通道开放调节的下游机制之间的耦合，而不是结构域开关本身。
Interdomain interactions between N-terminal and central domains serving as a "domain switch" are believed to be essential to the functional regulation of the skeletal muscle ryanodine receptor-1 Ca2+ channel. Mutational destabilization of the domain switch in malignant hyperthermia (MH), a genetic sensitivity to volatile anesthetics, causes functional instability of the channel. Dantrolene, a drug used to treat MH, binds to a region within this proposed domain switch. To explore its mechanism of action, the effect of dantrolene on MH-like channel activation by the synthetic domain peptide DP4 or anti-DP4 antibody was examined. A fluorescence probe, methylcoumarin acetate, was covalently attached to the domain switch using DP4 as a delivery vehicle. The magnitude of domain unzipping was determined from the accessibility of methylcoumarin acetate to a macromolecular fluorescence quencher. The Stern-Volmer quenching constant (K-Q) increased with the addition of DP4 or anti-DP4 antibody. This increase was reversed by dantrolene at both 37 and 22degreesC and was unaffected by calmodulin. [H-3]Ryanodine binding to the sarcoplasmic reticulum and activation of sarcoplasmic reticulum Ca2+ release, both measures of channel activation, were enhanced by DP4. These activities were inhibited by dantrolene at 37degreesC, yet required the presence of calmodulin at 22degreesC. These results suggest that the mechanism of action of dantrolene involves stabilization of domain-domain interactions within the domain switch, preventing domain unzipping-induced channel dysfunction. We suggest that temperature and calmodulin primarily affect the coupling between the domain switch and the downstream mechanism of regulation of Ca2+ channel opening rather than the domain switch itself.