SUBSPECIALIZATION OF CARDIAC SARCOPLASMIC RETICULUM

描述(改编自摘要)：本申请描述研究 调控细胞内钙释放的关键蛋白的特性研究 心肌中的肌浆网(JSR)。四个SR蛋白将是 研究过的，在心里有丰富的表达，共同定位于 结合膜，并形成复合体。这四种蛋白质是连接素， 三聚氰胺、钙调素和兰尼定受体(RyR)。互动 稳定这种结合蛋白复合体的特征将被表征，并且 阐明了该复合体在调节钙释放中的作用。容金是 JSR中主要的钙调素结合蛋白。它是一个完整的膜 一种蛋白质，与三聚氰胺一起，似乎将钙调素锚定在 JSR膜。重组连接素在Sf21中的表达和纯化 昆虫细胞及其与钙调素、三聚氰胺和钙调素的结合作用 RyR进行了调查。将产生针对连接素的抗体 超微结构定位。连接素基因将被克隆，并 已定义内含子-外显子边界。Triadin是第二种钙调素结合蛋白 心脏JSR中的蛋白质，它与连接素同源，也与 RyR。心脏中存在三种Triadin亚型，它们将被表达和 纯净的。Triadin与连接素、钙调蛋白和RyR的相互作用 将被描述为。特定部位的抗体将用于 心脏各Triadin亚型的超微结构定位。 钙调素是JSR中主要的腔内钙结合蛋白。它 似乎通过与Ryanodine受体的相互作用锚定在Ryanodine受体上 Triadin和Junctin。钙调素与三聚氰胺和三聚氰胺结合的研究 连接蛋白将被本地化。RyR是钙释放通道，它 从交界处的管腔面与上方的复合体相关联 薄膜。与复合体相互作用的RyR的亚域将是 已确认身份。蛋白质复合体功能将通过生化手段进行研究 连接蛋白、三聚氰胺和钙调素与RyR联合重建的研究 脂质体；通过与RyR在Sf21昆虫中的共表达 细胞；以及通过在转基因小鼠中靶向过表达这些蛋白 红心。评估钙释放的研究将使用每一种方法进行 系统。最后，其他三类RyR监管网站， 定位于细胞质一侧的细胞膜，会被定位。 这些是钙调蛋白结合位点(S)、PK-A磷酸化位点和 钙解酶的裂解部位。这些研究的完成将增加我们的 对分子结构和蛋白质相互作用的理解 发生在连接膜上的。结构角色和功能角色 在控制心脏钙释放的几种基本蛋白质中， 已定义。

DESCRIPTION (Adapted from the abstract): This application describes studies on characterization of key proteins regulating Ca release from junctional sarcoplasmic reticulum (JSR) in cardiac muscle. Four SR proteins will be studied, which are abundantly expressed in heart, co-localize to the junctional membrane, and form a complex. These four proteins are junctin, triadin, calsequestrin, and the ryanodine receptor (RyR). The interactions stabilizing this junctional protein complex will be characterized and the role of the complex in regulating Ca release elucidated. Junctin is the major calsequestrin-binding protein in JSR. It is an integral membrane protein which, along with triadin, appears to anchor calsequestrin to the JSR membrane. Recombinant junctin will be expressed and purified from Sf21 insect cells and its binding interactions with calsequestrin, triadin, and the RyR investigated. Antibodies to junctin will be produced for ultrastructural localization. The junctin gene will be cloned and the intron-exon boundaries defined. Triadin is a second calsequestrin-binding protein in cardiac JSR, which is homologous to junctin, and also binds to the RyR. Three triadin isoforms exist in heart which will be expressed and purified. Triadin interactions with junctin, calsequestrin, and the RyR will be characterized. Site-specific antibodies will be used for ultrastructural localization of each triadin isoform in heart. Calsequestrin is the major intraluminal Ca-binding protein in JSR. It appears to be anchored to the ryanodine receptor via interactions with triadin and junctin. The studies of calsequestrin binding to triadin and junctin will be localized. The RyR is the Ca release channel, which associates with the complex above from the lumenal face of the junctional membrane. The subdomain of the RyR interacting with the complex will be identified. Protein complex function will be investigated by biochemical co-reconstruction of junctin, triadin, and calsequestrin with the RyR in liposomes; by co-expression of the proteins with the RyR in Sf21 insect cells; and by targeted overexpression of the proteins in transgenic mouse hearts. Studies assessing Ca release will be performed using each of these systems. Finally, three other categories of RyR regulatory sites, positioned on the cytoplasmic side of the membrane, will be localized. These are the calmodulin-binding site(s), the PK-A phosphorylation site, and the calpain-cleavage sites. Completion of these studies will increase our understanding of the molecular architecture and protein interactions occurring at the junctional membrane. The structural and functional roles of several fundamental proteins controlling Ca release in heart will be defined.