INITIATION OF CELL DEATH BY POTASSIUM CHANNEL BLOCK

描述（来自申请人的摘要）：程序性细胞死亡（细胞凋亡） 几乎在所有的生物体中都存在，用于去除多余的细胞 在正常的发展过程中。多种刺激均可诱导细胞死亡 程序和许多分子参与的过程已经确定。三 果蝇基因，reaper，grim，和hid，在一个细胞中触发凋亡细胞死亡。 许多不同的情况下，但他们的作用机制是未知的。 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K+通道的失活， 天然灭活颗粒和全长Reaper的浓度范围 蛋白质在非常低的浓度下导致稳定的K+通道阻断。在 本质上，这些分子似乎是作为稳定的K+通道失活 颗粒或细胞内K+通道毒素。相反，应用 全长Reaper蛋白质的Na+通道的细胞质侧导致 Na+通道失活率降低。这些观察结果导致了 以下假设：凋亡蛋白，通过增加K+ 通道失活或阻断和/或Na+通道失活减少，结果 严重的膜去极化和细胞死亡级联的激活。的 这里描述的实验旨在通过检查来验证这一假设。 野生型和突变型Reaper蛋白对K+和Na+通道的影响 活性、膜电位和细胞杀伤活性 PC 12细胞系。我们将确定是否Reaper诱导的K+通道阻滞（或Na+通道阻滞） 通道开放）、膜去极化和细胞死亡可以通过 Reaper不敏感K+通道的过表达，通过使用药理学方法， 阻断Na+通道的药物，和/或通过共表达其他凋亡的 抑制剂的Reaper和K+或Na+通道之间的物理相互作用 将使用以下方法确定经历凋亡性细胞死亡的细胞 免疫共沉淀分析。最后，野生型和突变型 感染上级宫颈细胞杀伤活性的Reaper表达 将检查神经节交感神经元，以确定我们提出的 细胞凋亡起始的机制对于原代神经元是正确的。这 应用集成了经典的分子遗传学和细胞生物学方法 用电生理学方法研究细胞死亡， 为细胞死亡的启动机制提供了新的见解， 有助于开发新的治疗策略， 预防或治疗神经退行性疾病。

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.