BIOLOGICAL ROLE OF INTRACELLULAR CA2+ BINDING PROTEINS

细胞内钙的变化代表着一种重要的信号 兴奋收缩和刺激-反应耦合。的目标是 我们的实验室一直致力于识别细胞内 钙结合蛋白，以了解钙结合蛋白的中介作用 细胞内钙信号。这项修订后的续期计划侧重于 67 kDa钙调蛋白(Annexin)的生理功能研究 六)。膜联蛋白是一个由七个结构上相关的钙- 依赖的磷脂结合蛋白。我们目前的结果 证明肌肉组织中含有最高浓度的 Annexin VI.抗Annexin VI与钙泵共定位， 提示与肌浆网(SR)有关。在……里面 此外，SR的分馏研究表明共提纯了 膜联蛋白VI与终端池。 我们提出了四个必要的具体目标来定义生理学 膜联蛋白VI的功能：(1)磷脂结合常数 将测定的钙以评价生理性的 调控的意义；(2)抗Annexin VI抗体将 与钙泵、钙调素和 兰诺定敏感的钙释放通道；(3)重构研究 将在平面脂质中进行HSR蛋白质和组分的研究 为了进一步解决Annexin VI的调节作用而进行双分子层 钙依赖的钙释放。光亲和标记研究 将用于鉴定分离的Annexin VI靶蛋白 以及(4)膜联蛋白VI的表达将是 通过使用渗透性的、稳定的反义来选择性地抑制 培养的小鼠肌肉细胞中的S寡核苷酸。表型 检查的是钙的收缩/松弛和动力学。 释放/摄取。膜联蛋白VI在骨骼中的功能研究进展 肌肉将通过相关的定位研究在这个 高度有序的组织与钙释放/摄取的调节 明确的细胞器，SR，并具有高度变化的 分化表型，收缩。我们的研究将提供 膜联蛋白VI在心肌梗死中的生理作用的确凿证据 维持细胞内钙动态平衡。

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.