Role of Ion Channels in Cell Death

离子通道在细胞死亡中的作用

• 批准号: 6690033
• 负责人: WILLIAM P SCHILLING
• 金额: $ 30.3万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-01-01 至 2005-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6690033
• 关键词: adenosine triphosphate; biological transport; calcium flux; calcium indicator; cations; cell death; cell growth regulation; cell membrane; computer data analysis; conformation; electrophysiology; fluorescence microscopy; homeostasis; intermolecular interaction; marine toxins; membrane channels; oxidative stress; protein structure function; purinergic receptor; single cell analysis; tissue /cell culture; voltage /patch clamp; western blottings

The long-range goal of this research is to understand the molecular mechanisms associated with cell death in cells of the blood and vasculature. Necrotic cell death, or oncosis, can result in vascular cells from oxidant stress which may arise following activation of neutophils, uptake of low-density lipoproteins, or ischemia-reperfusion injury. Although a role for Ca2+ in necrotic cell death is well established, the molecular link(s) between a rise in cytosolic free Ca2+ concentration ([Ca2+]i) and subsequent cell lysis remains unknown. In recent studies, we have focussed on understanding the interaction of the potent marine toxin, maitotoxin (MTX), with endogenous cellular pathways leading to cell lysis. MTX, at picomolar concentrations, activates Ca2+-permeable, nonselective cation channels (CaNSC) leading directly to a sustained elevation of [Ca2+li. A secondary response to MTX is the formation of aqueous "pores" subsequent to the elevation of [Ca2+]i, which appear to grow in size, and allow passage of vital dyes into the cell. MTX-activated channels and pores are found in all mammalian cells examined to date including aortic endothelial cells, HEK293 cells, THP-1 monocytes, skin fibroblasts, and BW5147.3 lymphoma cells. The ubiquitous pattern of MTX-induced effects and the apparent high affinity suggests a mechanism of cytotoxicity that is specific and highly conserved throughout evolution. Indeed, the effects of MTX are indistinguishable from activation of P2Z/P2X7 purinergic receptors. In this regard, our most recent studies have shown that MTX and P2Z/P2X7 receptor stimulation activate distinct channels, but a common cytolytic pore. We have termed this ubiquitous pore, the cytolytic/oncotic pore, or COP. Using a combination of electrophysiological, biochemical, and molecular biological approaches, the specific aims of this proposal will test several hypotheses concerning the structure, function and regulation of COP. The specific aims are to determine 1) the molecular mechanism(s) by which CaNSC activity is linked to activation of COP, 2) the role of CaNSC activity and COP in activation of oncosis and/or apoptosis, and 3) the role of CaNSC and COP in oxidant-induced cell death. The proposed studies will increase our understanding of the molecular mechanisms associated with oncosis and apoptosis, provide a detailed analysis of the initial events involved in pore formation, and have important implications for cellular responses in vascular disease models.

这项研究的远距离目标是了解血液和脉管系统细胞中与细胞死亡相关的分子机制。 坏死细胞死亡或肿瘤病可导致血管细胞受到氧化应激，这可能会在激活中粒细胞激活，低密度脂蛋白或缺血 - 再灌注损伤后出现。 尽管Ca2+在坏死细胞死亡中的作用已得到很好的确定，但胞质游离Ca2+浓度（[Ca2+] i）与随后的细胞裂解之间的分子联系仍然未知。 在最近的研究中，我们专注于了解有效的海洋毒素Maitotoxin（MTX）与内源性细胞途径的相互作用，导致细胞裂解。 MTX在皮摩尔浓度下激活Ca2+可渗透性的非选择性阳离子通道（CANSC），直接导致[Ca2+li的持续升高。 对MTX的次要响应是[Ca2+] I高升高后的水性“孔”的形成，它们似乎在大小上生长，并允许将重要染料传递到细胞中。 迄今为止，在所有检查的哺乳动物细胞中都发现了MTX激活的通道和孔，包括主动脉内皮细胞，HEK293细胞，THP-1单核细胞，皮肤成纤维细胞和BW5147.3淋巴瘤细胞。 MTX诱导的效应和明显的高亲和力的普遍模式表明了一种细胞毒性的机制，在整个进化过程中都是特异性且高度保守的。 实际上，MTX的作用与P2Z/P2X7嘌呤能受体的激活没有区别。 在这方面，我们的最新研究表明，MTX和P2Z/P2X7受体刺激激活了不同的通道，但是一种常见的细胞溶解孔。 我们称这种无处不在的毛孔，细胞溶解/肿瘤孔或COP。 使用电生理，生化和分子生物学方法的结合，该提案的具体目的将检验有关COP结构，功能和调节的几种假设。 具体目的是确定1）CANSC活性与COP激活有关的分子机制，2）CANSC活性和COP在激活肿瘤和/或凋亡激活中的作用，以及3）CANSC和COP在氧化剂诱导的细胞死亡中的作用。 拟议的研究将增加我们对与肿瘤和凋亡相关的分子机制的理解，对孔形成所涉及的初始事件进行详细分析，并对血管疾病模型中的细胞反应具有重要意义。