Role of Ion Channels in Cell Death

本研究的长期目标是了解与血液和血管细胞死亡相关的分子机制。坏死细胞死亡或肿瘤可导致血管细胞氧化应激，氧化应激可能由中性粒细胞激活、低密度脂蛋白摄取或缺血再灌注损伤引起。虽然Ca2+在坏死细胞死亡中的作用已经确定，但胞质游离Ca2+浓度（[Ca2+]i）升高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，我们专注于了解强效海洋毒素maitotoxin （MTX）与内源性细胞途径导致细胞裂解的相互作用。在皮摩尔浓度下，MTX激活Ca2+渗透性，非选择性阳离子通道（CaNSC），直接导致[Ca2+li]的持续升高。MTX的第二个反应是在[Ca2+]i升高后形成水“孔”，其大小似乎在增长，并允许重要的染料进入细胞。迄今为止，在所有哺乳动物细胞中都发现了mtx激活的通道和毛孔，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诱导作用的普遍模式和明显的高亲和力表明细胞毒性机制在整个进化过程中是特异性和高度保守的。事实上，MTX的作用与P2Z/P2X7嘌呤能受体的激活是无法区分的。在这方面，我们最近的研究表明，MTX和P2Z/P2X7受体刺激激活不同的通道，但有一个共同的细胞溶解孔。我们将这种普遍存在的孔称为细胞溶解孔（COP）。结合电生理学、生物化学和分子生物学的方法，本提案的具体目标将测试关于COP的结构、功能和调节的几个假设。具体目的是确定1)CaNSC活性与COP激活相关的分子机制，2)CaNSC活性和COP在肿瘤和/或细胞凋亡激活中的作用，以及3)CaNSC和COP在氧化诱导的细胞死亡中的作用。这些研究将增加我们对肿瘤和细胞凋亡相关分子机制的理解，为孔隙形成的初始事件提供详细的分析，并对血管疾病模型中的细胞反应具有重要意义。

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.