Collaborative Project: Development of the Triad Junction in Skeletal Muscle

该项目的长期目标是了解肌肉细胞三联体连接形成和功能的分子机制。 这是质膜和肌浆网 (SR) 膜之间的连接处，被认为在耦合兴奋和肌肉收缩方面发挥着关键作用。 这项研究将建立三联体连接的一个关键元件，即胚胎和新生鸡骨骼肌中的“脚”蛋白的发育历史，以便深入了解该蛋白在连接形成、肌肉形态发生和 SR 膜生物发生中的作用。 植物生物碱兰尼碱与“足”蛋白特异性结合；因此，该化合物是该蛋白质的有用实验探针。 将检查“脚”或兰尼碱结合蛋白（RBP）出现的发育时间过程以及其结合兰尼碱和改变SR膜的钙渗透性的能力的表现。 “脚”蛋白的出现将通过特异性氚化兰尼碱结合来测量，并使用 SDS-PAGE 和 RBP 特异性抗体检测 400-500 kDa RBP。 “脚”蛋白功能的出现将通过测量细胞内钙瞬变和收缩活性来测定。 这些事件将与胚胎和新生雏鸡胸肌中成熟肌肉形态和 SR 功能的发育以及使用电子显微镜和免疫细胞化学技术可视化的胚胎肌肉原代培养物相关。 这项研究的结果应该大大有助于我们理解横纹肌细胞表面膜的激发触发细胞内隔室（肌浆网）钙释放的机制，这是启动肌肉收缩的关键步骤。

The long term goals of this project are to understand the molecular mechanisms underlying the formation and function of the triad junction in muscle cells. This is a junction between plasma and sarcoplasmic reticulum (SR) membranes that is thought to have a critical role in coupling excitation and muscle contraction. This research will establish the developmental history of a key element of the triad junction, the "foot" protein in embryonic and neonatal chick skeletal muscle, in order to gain insight into the role of this protein in junction formation, muscle morphogenesis and the biogenesis of SR membranes. The plant alkaloid, ryanodine, binds specifically to the "foot" protein; this compound is, thus, a useful experimental probe of this protein. The developmental time course of the appearance of the "foot" or ryanodine-binding protein (RBP) and of the manifestation of its ability to bind ryanodine and to alter the calcium permeability of SR membranes will be examined. The appearance of the "foot" protein will be measured by specific tritiated ryanodine binding, and detection of the 400-500 kDa RBP using SDS-PAGE and RBP specific antibodies. The appearance of "foot" protein function will be assayed by measurement of intracellular calcium transients and contractile activity. These events will be correlated with the development of a mature muscle morphology and SR function in pectoral muscle from embryonic and neonatal chicks and primary cultures of embryonic muscle as visualized using electron microscopy and immunocytochemical techniques. The results of this research should contribute substantially to our understanding of the mechanism by which excitation of the surface membrane of a striated muscle cell triggers calcium release from an intracellular compartment, the sarcoplasmic reticulum, as the key step in initiation of muscle contraction.