Co-expression in CHO cells of two muscle proteins involved in excitation-contraction coupling
Co-expression in CHO cells of two muscle proteins involved in excitation-contraction coupling
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两种参与兴奋-收缩耦合的肌肉蛋白在 CHO 细胞中共表达
DOI：
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发表时间：
1995
影响因子：
2.7
通讯作者：
C. Franzini
中科院分区：
文献类型：
--
作者：
H. Takekura;H. Takeshima;S. Nishimura;Masami Takahashi;T. Tanabe;V. Flockerzi;F. Hofmann;C. Franzini
摘要兰尼碱受体和二氢吡啶受体位于肌浆网与骨骼肌表面膜或横管之间的连接处，彼此相对。 Ryanodine 受体是肌浆网的钙释放通道，其细胞质结构域形成脚，将肌浆网与横管连接起来。二氢吡啶受体是L型钙通道，充当兴奋-收缩耦合的电压传感器：它们感知表面膜和横管去极化并诱导肌浆网释放通道的开放。在骨骼肌中，兰尼碱受体以广泛的阵列排列，二氢吡啶受体分为四联体，四联体又与兰尼碱受体的四个亚基相关。这种配置允许两组分子之间直接相互作用。除了α2、β和γ亚基的质粒之外，还用骨骼肌兰尼碱受体和骨骼二氢吡啶受体的质粒，或可以在功能上替代骨骼二氢吡啶受体的骨骼-心脏二氢吡啶受体嵌合体（CSk3）稳定转化CHO细胞。 RNA 印迹杂交对所有组分均给出阳性结果。免疫印迹、兰尼碱结合、电子显微镜和咖啡因暴露表明，表达的兰尼碱受体形成功能性四聚体通道，其正确插入内质网膜，并形成与骨骼肌中具有相同间距的广泛阵列。由于阵列的形成不需要二氢吡啶受体的共表达，因此我们得出结论，自聚集是兰尼定受体的独立特性。所有表达二氢吡啶受体的克隆均表现出与二氢吡啶的高亲和力结合，并用针对二氢吡啶受体的抗体进行免疫标记。 CSk3 克隆表面膜中存在具有快速动力学的钙电流和二氢吡啶受体的免疫标记，表明 CSk3-二氢吡啶受体适当地靶向细胞的质膜。然而，表达的骨骼型二氢吡啶受体仍然大部分位于核周膜内。在共表达功能性二氢吡啶受体和兰尼碱受体的细胞中，足内质网元件和表面膜之间不形成连接，并且二氢吡啶受体不组装成四联体。二氢吡啶受体和兰尼碱受体之间的分离并非CHO细胞所独有，在心肌、无脊椎动物的肌肉中以及在某些条件下在骨骼肌中也发现了这种现象。我们认为，在共转染的 CHO 细胞中未能形成连接可能是由于缺乏内质网与表面膜的初始对接或维持二氢吡啶受体和兰尼定受体之间的相互作用所必需的必需蛋白质。我们还得出结论，四分体的形成需要二氢吡啶受体和兰尼定受体之间的密切相互作用。
SummaryRyanodine receptors and dihydropyridine receptors are located opposite each other at the junctions between sarcoplasmic reticulum and either the surface membrane or the transverse tubules in skeletal muscle. Ryanodine receptors are the calcium release channels of the sarcoplasmic reticulum and their cytoplasmic domains form the feet, connecting sarcoplasmic reticulum to transverse tubules. Dihydropyridine receptors are L-type calcium channels that act as the voltage sensors of excitation-contraction coupling: they sense surface membrane and tranverse tubule depolarization and induce opening of the sarcoplasmic reticulum release channels. In skeletal muscle, ryanodine receptors are arranged in extensive arrays and dihydropyridine receptors are grouped into tetrads, which in turn are associated with the four subunits of ryanodine receptors. The disposition allows for a direct interaction between the two sets of molecules.CHO cells were stably transformed with plasmids for skeletal muscle ryanodine receptors and either the skeletal dihydropyridine receptor, or a skeletal-cardiac dihydropyridine receptor chimera (CSk3) which can functionally substitute for the skeletal dihydropyridine receptor, in addition to plasmids for the α2, β and γ subunits. RNA blot hybridization gave positive results for all components. Immunoblots, ryanodine binding, electron microscopy and exposure to caffeine show that the expressed ryanodine receptors forms functional tetrameric channels, which are correctly inserted into the endoplasmic reticulum membrane, and form extensive arrays with the same spacings as in skeletal muscle. Since formation of arrays does not require coexpression of dihydropyridine receptors, we conclude that self-aggregation is an independent property of ryanodine receptors. All dihydropyridine receptor-expressing clones show high affinity binding for dihydropyridine and immunolabelling with antibodies against dihydropyridine receptor. The presence of calcium currents with fast kinetics and immunolabelling for dihydropyridine receptors in the surface membrane of CSk3 clones indicate that CSk3-dihydropyridine receptors are appropriately targeted to the cell's plasmalemma. The expressed skeletal-type dihydropyridine receptors, however, remain mostly located within perinuclear membranes. In cells coexpressing functional dihydropyridine receptors and ryanodine receptors, no junctions between feet-bearing endoplasmic reticulum elements and surface membrane are formed, and dihydropyridine receptors do not assemble into tetrads. A separation between dihydropyridine receptors and ryanodine receptors is not unique to CHO cells, but is found also in cardiac muscle, in muscles of invertebrates and, under certain conditions, in skeletal muscle. We suggest that failure to form junctions in co-transfected CHO cell may be due to lack of an essential protein necessary either for the initial docking of the endoplasmic reticulum to the surface membrane or for maintaining the interaction between dihydropyridine receptors and ryanodine receptors. We also conclude that formation of tetrads requires a close interaction between dihydropyridine receptors and ryanodine receptors.
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DOI：
10.1016/s0021-9258(18)54728-1
发表时间：
1991-11
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
X. Wei;E. Perez-Reyes;A. E. Lacerda;G. Schuster;A. Brown;L. Birnbaumer
通讯作者：
X. Wei;E. Perez-Reyes;A. E. Lacerda;G. Schuster;A. Brown;L. Birnbaumer
DOI：
10.1016/s0021-9258(19)75701-9
发表时间：
1987-02
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
M. Inui;A. Saito;S. Fleischer
通讯作者：
M. Inui;A. Saito;S. Fleischer
通过二氢吡啶受体 cDNA 恢复发育不良肌管中的连接四分体。
影响因子：
3.4
作者：
Takekura,H;Bennett,L;Tanabe,T;Beam,KG;Franzini-Armstrong,C
通讯作者：
Franzini-Armstrong,C
小鼠 L 细胞中表达的兔心脏 L 型 Ca2 通道 α 1 亚基的 β 亚基调节的表征。
影响因子：
3.5
作者：
Lory,P;Varadi,G;Slish,DF;Varadi,M;Schwartz,A
通讯作者：
Schwartz,A
来自骨骼肌肌浆网的纯化兰尼碱受体是钙释放通道的 Ca2 渗透孔。
DOI：
--
发表时间：
1987
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Imagawa,T;Smith,JS;Coronado,R;Campbell,KP
通讯作者：
Campbell,KP