INITIATION OF CELL DEATH BY POTASSIUM CHANNEL BLOCK

描述(摘自申请者摘要)：程序性细胞死亡(细胞凋亡) 几乎存在于所有生物体中，用来去除多余的细胞 在正常发育过程中。多种刺激都能诱导细胞死亡。 程序和许多参与这一过程的分子已经被识别出来。三 果蝇基因，REPAR，GRIM和HID，在一个 有许多不同的背景，但它们的作用机制尚不清楚。 我们观察到来自Reaper和Grim的N-末端合成肽 蛋白质诱导电压门控性钾通道失活 作为天然灭活颗粒和全长收割机的浓度范围 蛋白质在很低的浓度下会导致稳定的钾通道阻断。在……里面 本质上，这些分子似乎起着稳定的K通道失活的作用 颗粒或细胞内K通道毒素。相反，应用 全长的收割机蛋白到钠通道的细胞质一侧导致 降低钠通道失活率。这些观察结果导致了 以下假设：细胞凋亡蛋白，或者通过增加K 通道失活或阻断和/或减少钠通道失活，结果 在严重的膜去极化和激活的细胞死亡级联。这个 这里描述的实验旨在通过检验 野生型和突变型Reaper蛋白对钾、钠通道的影响 稳定转染细胞的活性、膜电位和细胞杀伤活性 PC12细胞株。我们将确定死神诱导的K通道阻断(或Na 通道开放)、膜去极化和细胞死亡可以通过 用药理学方法过量表达收割机不敏感的K通道 阻断钠通道和/或通过与其他凋亡细胞共表达的药物 抑制剂。收割机与钾、钠通道的物理相互作用 正在经历细胞凋亡性死亡的细胞将使用 免疫共沉淀法。最后，野生型和突变型的影响 Reaper在感染上颈椎组织中的表达及其对细胞杀伤活性的影响 将对神经节交感神经元进行检查，以确定我们提议的 对于原代神经元来说，启动细胞凋亡的机制是正确的。这 结合经典分子遗传学和细胞生物学方法的应用 用预期的电生理学方法研究细胞死亡 为启动细胞死亡的机制提供新的见解，并可能 为开发新的治疗策略做出贡献 神经退行性疾病的预防或治疗。

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.