REGULATION OF ION CHANNELS BY APOPTOSIS TRIGGER PROTEINS

细胞凋亡或程序性细胞死亡可由多种刺激诱导。细胞凋亡被认为参与了许多生理和临床现象，包括缺血性损伤、多种形式的癌症和神经退行性疾病，如阿尔茨海默病、肌萎缩侧索硬化症和一些朊病毒相关疾病。许多诱导细胞凋亡的刺激物和基因产物已被确定。然而，这些刺激触发细胞死亡程序的分子机制却知之甚少。这里提出的研究计划将集中于凋亡触发基因reaper如何促进电兴奋细胞的细胞死亡。死神基因在细胞死亡程序开始前1至2小时在注定要经历凋亡的细胞中表达。通过对Reaper蛋白n端与A型电压依赖性钾离子通道（ShB）的序列比较，表明Reaper蛋白可以作为一个失活球阻断钾离子通道。我们的初步结果确实表明，全长Reaper蛋白通过诱导非常稳定的失活来阻断电压依赖性钾通道。我们的初步结果还表明，死神减缓了电压依赖性钠通道的失活过程。拟议的研究计划将研究死神蛋白如何改变电压依赖性离子通道的功能特性以促进细胞凋亡。中心假设是Reaper改变质膜离子通道的特性以促进去极化，而这种去极化促进细胞凋亡。我们将利用非洲爪蟾卵母细胞中异源表达的离子通道以及原生哺乳动物通道来验证这一假设。我们将研究Reaper诱导表达如何影响哺乳动物细胞中的离子通道特性和膜电位。这项研究的预期结果将确定一些细胞凋亡触发蛋白如何促进细胞死亡，结果可能有助于设计治疗干预措施来治疗涉及细胞凋亡的疾病。

Apoptosis or programmed cell death can be induced by a variety of stimuli. Apoptosis is considered to be involved in many physiological and clinical phenomena, including ischemic injury, many forms of cancer, and neurodegenerative diseases, such as Alzheimer's disease, amyotrophic lateral sclerosis, and some prion-related disorders. Many apoptosis-inducing stimuli and gene products have been identified. The molecular mechanisms by which these stimuli trigger the cell-death program are, however, only poorly understood. The research program proposed here will focus on how an apoptosis-trigger gene, reaper, promotes cell death in electrically excitable cells. The reaper gene is expressed in cells destined to undergo apoptosis 1 to 2 hours before the cell death program starts. A sequence comparison of the N-termini of Reaper and a "A-type" voltage-dependent potassium channel (ShB) shows that the Reaper protein could work as an inactivation ball to block potassium channels. Our preliminary results indeed show that the full-length Reaper protein blocks voltage-dependent potassium channels by inducing very stable inactivation. Our preliminary results also show that Reaper slows down the inactivation process of voltage-dependent sodium channels. The proposed research program will examine how the Reaper protein alters functional properties of voltage-dependent ion channels to promote apoptosis. The central hypothesis is that Reaper alters properties of the plasma membrane ion channels to promote depolarization and that this depolarization facilitates apoptosis. We will test this hypothesis using heterologously expressed ion channels in Xenopus oocytes as well as native mammalian channels. We will examine how induced expression Reaper affects the ion channel properties and membrane potential in mammalian cells. The results expected from the proposed research will establish how some apoptosis-trigger proteins promote cell-death and the results may be useful in designing therapeutic interventions to treat the diseases that involve apoptosis.