REGULATION OF THE CARDIAC NA+/CA+2 EXCHANGER

在这个项目中，申请者将调查 转基因中国仓鼠卵巢的钠/钙交换活性 永久表达野生型心肌钠/钙的细胞 定点突变产生的交换子或突变型交换子。 中心假说是完整细胞中的交换活动是 在三个不同的复杂性水平上进行调控：(1)通过动力学 胞浆(Ca~(2+))变化引起的改变，(2)通过 与细胞骨架的相互作用，以及(3)通过与 钙离子封存细胞器。申请者将描述 胞浆(Na)、(Ca)和pH对交换活性的调节 通过测量交换介导的Ba2在经 离子载体革兰西丁和离子霉素。申请者还将 检测细胞骨架变化对交换活性的影响 由磷酸酶抑制剂Calyculin A和渗透压诱导 收缩，特别强调中间细丝的作用 在回应中。最后，申请者将使用选择性抑制剂和 检测钙摄取作用的CA2引导/耗竭方案 并通过内质网和其他钙离子的隔离释放 激活或停用交换活动的细胞器。

In this project, the applicant will investigate the regulation of Na+/Ca2+ exchange activity in transfected Chinese hamster ovary cells that permanently express either the wild-type cardiac Na+/Ca2+ exchanger or mutant exchangers created by site-directed mutagenesis. The cental hypothesis is that exchange activity in intact cells is regulated at three different levels of complexity: (1) throughkinetic alterations induced by changes in cytosolic (Ca2+), (2) through interactions with the cytoskeleton, and (3) through interactions with Ca2+ sequestering organelles. The applicant will characterize the regulation of exchange activity by cytosolic (Na+), (Ca2+), and pH by measuring exchange-mediated Ba2+ influx in cells treated with the ionophores gramicidin and ionomycin. The applicant will also examine the influence of exchange activity of cytoskeletal changes induced by the phosphatase inhibitor calyculin A, and by osmotic shrinkage, with special emphasis on the role of intermediate filaments in the response. Finally, the applicant will use selective inhibitors and Ca2+ leading/depletion protocols to examine the role of Ca2+ uptake and release by the endoplasmic reticulum and otehr Ca2+ sequestering organelles in activating or deactivating exchange activity.