INITIATION OF CELL DEATH BY POTASSIUM CHANNEL BLOCK

钾通道阻滞引发细胞死亡

基本信息

批准号：
6285877
负责人：
Linda E Iverson
金额：
$ 26.25万
依托单位：
BECKMAN RESEARCH INSTITUTE/CITY OF HOPE
依托单位国家：
美国
项目类别：
财政年份：
2000
资助国家：
美国
起止时间：
2000-09-30 至 2005-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6285877
关键词：
Drosophilidae PC12 cells Xenopus oocyte apoptosis immunoprecipitation ion channel blocker membrane potentials neurotoxins potassium channel sodium channel synthetic peptide transfection voltage gated channel yeast two hybrid system

项目摘要

DESCRIPTION (From the Applicant's Abstract): Programmed cell death (apoptosis) occurs in virtually all organisms and is used to remove superfluous cells during normal development. A variety of stimuli can induce the cell death program and many molecules involved in the process have been identified. Three Drosophila genes, reaper, grim, and hid, trigger apoptotic cell death in a number of different contexts, yet the mechanism by which they act is unknown. We observed that N-terminal synthetic peptides derived from Reaper and Grim proteins induce inactivation of voltage-gated K+ channels in the same concentration range as the native inactivation particle and full-length Reaper protein results in stable K+ channel block at very low concentration. In essence, these molecules appear to be acting as stable K+ channel inactivation particles or intracellular K+ channel toxins. Conversely, application of full-length Reaper protein to the cytoplasmic side of Na+ channels leads to a decreased rate of Na+ channel inactivation. These observations have led to the following hypothesis: the apoptosis proteins, either through increased K+ channel inactivation or block and/or decreased Na+ channel inactivation, result in severe membrane depolarization and activation of the cell death cascade. The experiments described here are designed to test this hypothesis by examining the effects of wild-type and mutant Reaper proteins on K+ and Na+ channel activity, membrane potential, and cell killing activity in stably transfected PC12 cell lines. We will determine if Reaper-induced K+ channel block (or Na+ channel opening), membrane depolarization, and cell death can be prevented by over-expression of a Reaper-insensitive K+ channel, by using pharmacological agents that block Na+ channels, and/or by co-expression of other apoptotic inhibitors. Physical interactions between Reaper and K+ or Na+ channels in cells undergoing apoptotic cell death will be determined using co-immunoprecipitation assays. Finally, the effects of wild-type and mutant Reaper expression on cell killing activity in infected superior cervical ganglion sympathetic neurons will be examined to determine if our proposed mechanism for the initiation of apoptosis is true for primary neurons. This application integrates classical molecular genetic and cell biological methods for studying cell death with an electrophysiological approach that is expected to provide new insights into mechanisms of initiation of cell death and may contribute to the development of novel therapeutic strategies for the prevention or treatment of neurodegenerative disorders.

描述（来自申请人的摘要）：程序性细胞死亡（细胞凋亡）几乎存在于所有生物体中，用于去除多余的细胞正常发育过程中。多种刺激可引起细胞死亡程序和参与该过程的许多分子已被识别。三果蝇基因“reaper”、“grim”和“hid”会引发细胞凋亡许多不同的上下文，但它们的作用机制尚不清楚。我们观察到源自 Reaper 和 Grim 的 N 端合成肽蛋白质诱导电压门控 K+ 通道失活天然灭活颗粒和全长 Reaper 的浓度范围蛋白质在极低浓度下会产生稳定的 K+ 通道阻断。在本质上，这些分子似乎起到稳定 K+ 通道失活的作用颗粒或细胞内 K+ 通道毒素。相反，应用将全长 Reaper 蛋白连接到 Na+ 通道的细胞质侧会导致 Na+通道失活率降低。这些观察结果导致以下假设：凋亡蛋白，或者通过增加 K+ 通道失活或阻断和/或减少 Na+ 通道失活，结果严重的膜去极化和细胞死亡级联的激活。这这里描述的实验旨在通过检查来检验这一假设野生型和突变型 Reaper 蛋白对 K+ 和 Na+ 通道的影响稳定转染细胞的活性、膜电位和细胞杀伤活性 PC12细胞系。我们将确定收割者是否诱导 K+ 通道阻断（或 Na+ 通道打开）、膜去极化和细胞死亡可以通过以下方式预防：通过使用药理学方法过度表达对 Reaper 不敏感的 K+ 通道阻断 Na+ 通道的药物，和/或通过其他细胞凋亡的共表达抑制剂。 Reaper 与 K+ 或 Na+ 通道之间的物理相互作用经历细胞凋亡的细胞将使用以下方法确定免疫共沉淀分析。最后，野生型和突变型的影响 Reaper 表达对受感染上宫颈细胞杀伤活性的影响将检查神经节交感神经元以确定我们提出的是否细胞凋亡的启动机制对于初级神经元来说是正确的。这应用整合了经典的分子遗传学和细胞生物学方法用于通过预期的电生理学方法研究细胞死亡为细胞死亡的启动机制提供新的见解，并可能为开发新的治疗策略做出贡献预防或治疗神经退行性疾病。