BIOLOGICAL ROLE OF INTRACELLULAR CA2+ BINDING PROTEINS

细胞内钙离子的变化代表了一个重要的信号， 兴奋收缩和刺激-反应耦合。 的目标 我们的实验室一直致力于识别细胞内 钙结合蛋白，以了解介导的 细胞内钙信号。 此次更新的重点是 阐明了67 kDa钙调蛋白（膜联蛋白）的生理功能 VI）。 膜联蛋白是一个由七种结构相关的钙- 依赖磷脂结合蛋白。 我们目前的成果 表明肌肉组织中 膜联蛋白VI 抗膜联蛋白VI与钙泵共定位， 提示与肌浆网（SR）有关。 在 此外，SR的分馏研究表明， 具有末端池的膜联蛋白VI。 我们提出了四个必要的具体目标来定义生理 膜联蛋白VI的功能：（1）磷脂和 将测定钙，以评价生理 调节的意义;（2）抗膜联蛋白VI抗体将被 与钙泵、钙螯合蛋白和 （3）重组研究 的HSR蛋白质和馏分将进行，并在平面脂质 为了进一步解决膜联蛋白VI的调节作用， 钙依赖性钙释放。 光亲和标记研究 将用于鉴定分离的膜联蛋白VI靶蛋白， 末端池;和（4）膜联蛋白VI的表达将是 选择性抑制通过使用渗透性，稳定的反义 培养的小鼠肌肉细胞中的RNA S-寡核苷酸。 表型 将检查收缩/舒张速率和钙离子动力学 释放/摄取。 膜联蛋白VI在骨骼肌中的作用 肌肉将通过相关的定位研究获得， 高度有序的组织调节钙的释放/吸收 明确的细胞器，SR，并在一个高度变化， 分化表型，收缩。 我们的研究将提供 膜联蛋白VI的生理作用的明确证据， 维持细胞内钙稳态。

Changes in intracellular calcium represent an essential signal in excitation contraction and stimulus-response coupling. The objective of our laboratory has been directed towards identifying intracellular calcium-binding proteins in order to understand the mediation of the intracellular calcium signal. This revised renewal is focused towards elucidating the physiological function of the 67 kDa calcimedin (annexin VI). The annexins are a family of seven structurally related calcium- dependent phospholipid binding proteins. Our current results demonstrate that muscle tissues have the highest concentrations of annexin VI. Anti-annexin VI co-localizes with the calcium-pump, suggesting an association with the sarcoplasmic reticulum (SR). In addition, fractionation studies of SR demonstrate co-purifiiation of annexin VI with terminal cisternae. We propose four specific aims necessary to define the physiological function of annexin VI: (1) Binding constants for phospholipids and calcium will be determined in order to to evaluate the physiological significance of regulation; (2) Anti-annexin VI antibodies will be co-localized with the calcium-pump, calsequestrin and the ryanodine-sensitive calcium release channel; (3) Reconstitution studies of HSR proteins and fractions will be performed and in planar lipid bilayers in order to further resolve the regulatory role of annexin VI in calcium-dependent calcium release. Photoaffinity labeling studies will be employed to identify annexin VI target proteins in isolated terminal cisternae; and (4) The expression of annexin VI will be selectively depressed through the use of permeable, stable anti-sense RNA S-oligo nucleotides in cultured mouse muscle cells. The phenotypes examined will be rates of contraction/relaxation and dynamics of calcium release/uptake. Information on the function of annexin VI in skeletal muscle will be obtained by correlating localization studies in this highly ordered tissue with regulation of calcium release/uptake in a well-defined organelle, the SR, and with alteration in a highly differentiated phenotype, contraction. Our studies will provide definitive evidence for the physiological role of annexin VI in maintaining intracellular calcium homeostasis.