Collaborative Project: Development of the Triad Junction in Skeletal Muscle

该项目的长期目标是了解肌细胞中三联结形成和功能的分子机制。这是血浆和肌浆网（SR）膜之间的连接，被认为在耦合兴奋和肌肉收缩中起关键作用。本研究将建立三角连接的关键元件“脚”蛋白在胚胎和新生鸡骨骼肌中的发育历史，以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，ryanodine，专门与“脚”蛋白结合；因此，这种化合物是这种蛋白质的一种有用的实验探针。“足”或RBP结合蛋白（RBP）的出现及其结合ryanodine和改变SR膜钙通透性的能力的表现的发育时间过程将被检查。“脚”蛋白的外观将通过特异性氚化ryanodine结合来测量，并使用SDS-PAGE和RBP特异性抗体检测400-500 kDa的RBP。“足”蛋白功能的外观将通过测量细胞内钙瞬态和收缩活性来测定。通过电子显微镜和免疫细胞化学技术观察，这些事件将与胚胎和新生雏鸡的成熟肌肉形态和胸肌SR功能的发展以及胚胎肌肉的原代培养相关。这项研究的结果将大大有助于我们理解横纹肌细胞表面膜的兴奋触发钙从细胞内腔室（肌浆网）释放的机制，这是开始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.