Ohkura M, Furukawa K, Fujimori H, Kuruma A, Kawano S, Hiraoka M, Kuniyasu A, Nakayama H, Ohizumi Y
Department of Pharmaceutical Molecular Biology, Faculty of Pharmaceutical Sciences, Tohoku University, Sendai, Japan.
Biochemistry. 1998 Sep 15;37(37):12987-93. doi: 10.1021/bi972803d.
Triadin, a calsequestrin-anchoring transmembrane protein of the sarcoplasmic reticulum (SR), was successfully purified from the heavy fraction of SR (HSR) of rabbit skeletal muscle with an anti-triadin immunoaffinity column. Since depletion of triadin from solubilized HSR with the column increased the [3H]ryanodine binding activity, we tested a possibility of triadin for a negative regulator of the ryanodine receptor/Ca2+ release channel (RyR). Purified triadin not only inhibited [3H]ryanodine binding to the solubilized HSR but also reduced openings of purified RyR incorporated into the planar lipid bilayers. On the other hand, calsequestrin, an endogenous activator of RyR [Kawasaki and Kasai (1994) Biochem. Biophys. Res. Commun. 199, 1120-1127; Ohkura et al. (1995) Can. J. Physiol. Pharmacol. 73, 1181-1185] potentiated [3H]ryanodine binding to the solubilized HSR. Ca2+ dependency of [3H]ryanodine binding to the solubilized HSR was reduced by triadin, whereas that was enhanced by calsequestrin. Interestingly, [3H]ryanodine binding to the solubilized HSR potentiated by calsequestrin was reduced by triadin. Immunostaining with anti-triadin antibody proved that calsequestrin inhibited the formation of oligomeric structure of triadin. These results suggest that triadin inhibits the RyR activity and that RyR is regulated by both triadin and calsequestrin, probably through an interaction between them. In this paper, triadin has been first demonstrated to have an inhibitory role in the regulatory mechanism of the RyR.
肌集钙蛋白结合蛋白是肌浆网（SR）的一种跨膜蛋白，通过抗肌集钙蛋白结合蛋白免疫亲和柱，成功地从兔骨骼肌肌浆网重组分（HSR）中纯化得到。由于用该柱从溶解的HSR中去除肌集钙蛋白结合蛋白会增加[³H]ryanodine结合活性，我们测试了肌集钙蛋白结合蛋白作为ryanodine受体/Ca²⁺释放通道（RyR）负调节因子的可能性。纯化的肌集钙蛋白结合蛋白不仅抑制[³H]ryanodine与溶解的HSR结合，还减少了整合到平面脂质双分子层中的纯化RyR的开放。另一方面，肌集钙蛋白，一种RyR的内源性激活剂[川崎和笠井（1994年）生物化学与生物物理学研究通讯199，1120 - 1127；大仓等人（1995年）加拿大生理学与药理学杂志73，1181 - 1185]增强了[³H]ryanodine与溶解的HSR的结合。肌集钙蛋白结合蛋白降低了[³H]ryanodine与溶解的HSR结合的Ca²⁺依赖性，而肌集钙蛋白则增强了这种依赖性。有趣的是，肌集钙蛋白增强的[³H]ryanodine与溶解的HSR的结合被肌集钙蛋白结合蛋白降低。用抗肌集钙蛋白结合蛋白抗体进行免疫染色证明，肌集钙蛋白抑制了肌集钙蛋白结合蛋白寡聚体结构的形成。这些结果表明，肌集钙蛋白结合蛋白抑制RyR活性，并且RyR可能通过它们之间的相互作用受到肌集钙蛋白结合蛋白和肌集钙蛋白的共同调节。在本文中，首次证明肌集钙蛋白结合蛋白在RyR的调节机制中具有抑制作用。
