Collaborative Project: Development of the Triad Junction in Skeletal Muscle

本项目的长期目标是了解 的形成和功能的分子机制， 三联体连接处。 这是一个连接点， 质膜和肌浆网（SR）膜，被认为是 在连接兴奋和肌肉中起着关键作用， 收缩。 这项研究将建立发展 历史上的一个关键要素三位一体的交界处，“脚” 蛋白质在胚胎和新生鸡骨骼肌， 为了深入了解这种蛋白质在连接中的作用， 肌肉形态发生和SR的生物发生 膜。 植物生物碱，ryanodine，特异性地结合到 “脚”蛋白;这种化合物是，因此，一个有用的实验 这种蛋白质的探针。 发展的时间进程 出现"足"或ryanodine结合蛋白（RBP）， 它能结合ryanodine， 改变SR膜的钙渗透性。 "脚"蛋白的出现将通过特定的 氚标记的兰尼碱结合，以及400 - 500 kDa RBP的检测 使用SDS-PAGE和RBP特异性抗体。 的外观 “足”蛋白质功能将通过测量 细胞内钙瞬变和收缩活性。 这些 事件将与成熟肌肉的发育相关 胚胎和成年人胸肌的形态和SR功能 新生雏鸡和胚胎肌肉的原代培养物， 使用电子显微镜和免疫细胞化学观察 技术. 这项研究的结果将大大有助于 我们对这种机制的理解， 横纹肌细胞的表面膜触发钙离子 从细胞内释放，肌浆 网织，作为肌肉收缩开始的关键步骤。

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.