REGULATION OF THE CARDIAC NA+/CA+2 EXCHANGER

在本项目中，申请人将调查 转染中国仓鼠卵巢的Na+/Ca ~（2+）交换活性 永久表达野生型心脏Na+/Ca 2 + 交换剂或通过定点诱变产生的突变交换剂。 中心假设是完整细胞中的交换活动是 在三个不同的复杂性水平上调节：（1）通过动力学 细胞溶质（Ca 2+）变化引起的变化，（2）通过 与细胞骨架的相互作用，以及（3）通过与 Ca 2+螯合细胞器。 申请人将描述 通过胞质（Na+）、（Ca 2+）和pH调节交换活性 通过测量用以下物质处理的细胞中交换介导的Ba 2+流入， 离子载体短杆菌肽和离子霉素。 申请人还将 检查细胞骨架变化的交换活性的影响 由磷酸酶抑制剂calyculin A和渗透压 收缩率，特别强调中间长丝的作用 in the response响应. 最后，申请人将使用选择性抑制剂， Ca 2+引导/耗竭方案，以检查Ca 2+摄取的作用 通过内质网释放和其他Ca 2+螯合 细胞器激活或钝化交换活动。

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.