Role of Ion Channels in Cell Death

这项研究的长期目标是了解与血液和血管细胞中细胞死亡相关的分子机制。 坏死性细胞死亡或胀亡可导致血管细胞因氧化应激而死亡，氧化应激可在嗜中性粒细胞活化、低密度脂蛋白摄取或缺血-再灌注损伤后出现。 虽然Ca 2+在坏死性细胞死亡中的作用已得到充分证实，但胞质游离Ca 2+浓度（[Ca 2 +]i）升高与随后的细胞溶解之间的分子联系仍不清楚。 在最近的研究中，我们已经集中在了解的相互作用的有效的海洋毒素，maitotoxin（MTX），与内源性细胞途径导致细胞裂解。 MTX在皮摩尔浓度下激活Ca 2+可渗透的非选择性阳离子通道（CaNSC），直接导致[Ca 2 +] i的持续升高。 对MTX的次级反应是在[Ca 2 +]i升高之后形成水性“孔”，其大小似乎增加，并允许活体染料通过进入细胞。 MTX激活的通道和孔存在于迄今为止检查的所有哺乳动物细胞中，包括主动脉内皮细胞、HEK 293细胞、THP-1单核细胞、皮肤成纤维细胞和BW 5147.3淋巴瘤细胞。 MTX诱导效应的普遍存在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P2 Z/P2 X7嘌呤能受体的激活是不可区分的。 在这方面，我们最近的研究表明，MTX和P2 Z/P2 X7受体刺激激活不同的通道，但共同的溶细胞孔。 我们将这种普遍存在的孔称为溶细胞/溶瘤孔或COP。 使用电生理学，生物化学和分子生物学方法的组合，本提案的具体目标将测试几个假设的结构，功能和调节的COP。 具体目的是确定1）CaNSC活性与COP活化相关的分子机制，2）CaNSC活性和COP在胀亡和/或凋亡活化中的作用，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的理解与胀亡和凋亡的分子机制，提供了一个详细的分析，在孔的形成所涉及的初始事件，并在血管疾病模型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.