CYTOSOLIC MODULATION OF PLASMA MEMBRANE ION TRANSPORT

描述（根据申请人的抽象和具体目标改编）： 项目的重点是质膜钙泵（PMCa）。 PM Ca 泵是 去除细胞质钙的三种主要机制之一 因此常常在终止信号转导中发挥关键作用 许多细胞。 钙泵活性缺陷会导致钙增加， 随后 Na 和 K 发生变化。这些变化将改变关键 细胞质功能。 高血压、镰状细胞病、 缺血/再灌注损伤、兴奋性毒性、中风和阿尔茨海默病 显示细胞 Ca 稳态的改变。 PMCa 提供了多种 EP型泵结构功能研究的优点：与Na型泵不同 PMCa 是单个亚基。 与 SR Ca 泵相比， 蛋白质可以从细胞外介质中获取，这提供了 体细胞选择程序的优点。 这个应用程序是 重点关注细胞外释放步骤。 IC50 的变化 细胞外Ca 抑制可能是由于细胞外Ca 的变化所致 释放步骤、访问通道的改变或易位的改变 步骤或这些步骤的组合。 目标 1 是确定是否 细胞外 Ca 的释放通过高场通道发生。 目标2 是为了比较细胞外 CA 和 Mg 对雪貂心脏的抑制作用 当在爪蟾卵母细胞中表达时，钙泵与大鼠钙泵一起使用 确定更改哪些步骤以适应不同的灵敏度 到细胞外Ca。目标 3 是选择由以下方法产生的抗钙析出泵： PMCa cDNA 的随机突变（没有细胞的其他改变） 遗传学）。

DESCRIPTION (Adapted from applicant's abstract and specific aims): The focus of project is the plasma membrane Ca pump (PMCa). The PM Ca pump is one of the three primary mechanisms for the removal of cytoplasmic calcium and thus often plays a pivotal role in terminating signal transduction in many cells. Defects in Ca pump activity would lead to an increase in Ca, with subsequent changes in Na and K. These changes would alter key cytoplasmic functions. Hypertension, sickle cell disease, ischemia/reperfusion injury, excitotoxicity, stroke, and Alzheimer's disease show alterations in cellular Ca homeostasis. The PMCa offers several advantages for structure function studies of EP type pumps: Unlike the Na pump, PMCa is a single subunit. In contrast to the SR Ca pump, parts of the protein are accessible from the extracellular media and this offers advantages for somatic cell selection procedures. This application is focused on the extracellular release steps. Changes in the IC50 for extracellular Ca inhibition could result from changes in the extracellular release steps, alterations of an access channel or changes in translocation steps or a combination of these. Aim 1 is to determine whether extracellular Ca release occurs through a high field access channel. Aim 2 is to compare extracellular CA and Mg inhibition of the ferret cardiac calcium pump with rat calcium pump when expressed in xenopus oocytes and to determine which steps are altered to account for the different sensitivity to extracellular Ca. Aim 3 is to select Ca-out resistant pumps generated by random mutagenesis of the PMCa cDNA (without other alterations of cellular genetics).