Functional interaction of the cytoplasmic domain of triadin with the skeletal ryanodine receptor
Functional interaction of the cytoplasmic domain of triadin with the skeletal ryanodine receptor
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DOI：
10.1074/jbc.274.18.12278
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发表时间：
1999-04-30
影响因子：
4.8
通讯作者：
Ronjat, M
中科院分区：
文献类型：
--
作者：
Groh, S;Marty, I;Ronjat, M
Triadin 已被证明与肌浆网膜中的兰尼碱受体共定位。我们表明，用针对三联蛋白或兰尼碱受体的抗体对溶解的肌浆网膜进行免疫沉淀，导致兰尼碱受体和三联蛋白的免疫共沉淀，然后我们研究了三联蛋白的胞质结构域（残基 1-47）在控制肌浆网 Ca2+ 释放中的功能重要性，我们表明针对合成肽的抗体包括残基 2-17，诱导肌浆网囊泡释放 Ca2+ 的速率降低，以及掺入脂质双层中的兰尼碱受体 Ca2+ 通道的开放概率降低。使用表面等离子体共振光谱，我们定义了与纯化的兰尼碱受体相互作用的三联蛋白胞质部分的离散结构域（残基 18-46）。这种相互作用在低 Ca2+ 浓度（高达 pCa 5）时最佳，并通过增加钙浓度（IC50 为 300 μM）而受到抑制。该三联蛋白结构域与兰尼碱受体的直接分子相互作用通过重叠测定得到证实，并显示出诱导双层中纯化的 RyR 的 Ca2+ 通道活性的抑制。我们认为，这种相互作用在三联蛋白对兰尼碱受体的 Ca2+ 通道行为的控制中起着关键作用，因此可能代表骨骼肌兴奋-收缩耦合调节过程中的重要一步。
Triadin has been shown to co-localize with the ryanodine receptor in the sarcoplasmic reticulum membrane. We show that immunoprecipitation of solubilized sarcoplasmic reticulum membrane with antibodies directed against triadin or ryanodine receptor, leads to the coimmunoprecipitation of ryanodine receptor and triadin, We then investigated the functional importance of the cytoplasmic domain of triadin (residues 1-47) in the control of Ca2+ release from sarcoplasmic reticulum, We show that antibodies directed against a synthetic peptide encompassing residues 2-17, induce a decrease in the rate of Ca2+ release from sarcoplasmic reticulum vesicles as well as a decrease in the open probability of the ryanodine receptor Ca2+ channel incorporated in lipid bilayers, Using surface plasmon resonance spectroscopy, we defined a discrete domain (residues 18-46) of the cytoplasmic part of triadin interacting with the purified ryanodine receptor. This interaction is optimal at low Ca2+ concentration (up to pCa 5) and inhibited by increasing calcium concentration (IC50 of 300 mu M). The direct molecular interaction of this triadin domain with the ryanodine receptor was confirmed by overlay assay and shown to induce the inhibition of the Ca2+ channel activity of purified RyR in bilayer, We propose that this interaction plays a critical role in the control, by triadin, of the Ca2+ channel behavior of the ryanodine receptor and therefore may represent an important step in the regulation process of excitation-contraction coupling in skeletal muscle.