Regulation of PMCA Pump-Channels by Oxidant Stress

氧化应激对 PMCA 泵通道的调节

• 批准号: 7923951
• 负责人: WILLIAM P SCHILLING
• 金额: $ 40.42万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-01 至 2012-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7923951
• 关键词: ATP phosphohydrolase; Affinity; Alzheimer' s Disease; Architecture; Arteries; Atherosclerosis; Binding; Biological Aging; Blood Vessels; Ca(2+)-Transporting ATPase; Calcium; Cardiac Glycosides; Cations; Cell Death; Cell Line; Cell membrane; Cell physiology; Cells; Cysteine; Cytolysis; Development; Disease; Employee Strikes; Evaluation; Event; Evolution; Family; Glutathione; Glutathione Disulfide; Goals; Hand functions; Homeostasis; In Vitro; Insecta; Ion Channel; Ion Pumps; Ions; Kinetics; Lead; Link; Liposomes; Low-Density Lipoproteins; Marine Toxins; Mediating; Membrane; Modeling; Modification; Molecular; Molecular Conformation; Molecular Target; Monovalent Cations; Myocardial Infarction; Na(+)-K(+)-Exchanging ATPase; Nature; Necrosis; Ouabain; Oxidants; Oxidation-Reduction; Oxidative Stress; Pathway interactions; Permeability; Peroxidases; Physiological; Play; Preparation; Property; Proteins; Pump; RNA Interference; Regulation; Reperfusion Injury; Research Project Grants; Role; Signal Transduction; Stimulus; Stroke; Testing; Therapeutic Intervention; Toxin; Vascular Endothelial Cell; cell killing; density; in vivo; insight; interest; knockout gene; maitotoxin; man; marine organism; member; neuronal cell body; novel; operation; oxidant stress; oxidation; palytoxin; receptor; reconstitution; research study; response; tert-Butylhydroperoxide; uptake

Ion channels use the energy stored in ionic gradients to initiate rapid signaling events essential for the function of virtually every cell in the body. Although channel activation can occur in response to a variety of different stimuli, once open all channels share a common feature--they allow millions of ions per second to cross the membrane. Pump proteins on the other hand, use the energy in ATP to establish the ionic gradients necessary for channel function. Pumps generally move hundreds of ions per second, and hence their density in the membrane is by necessity much higher than that of channels. These striking differences led to the view that pumps and channels move ions across membranes by very different mechanisms. However, recent studies on the potent marine toxin, palytoxin (PTX), challenge this concept. PTX binds with picomolar affinity to the Na+,K+-ATPase (NKA) and converts the pump into a non-selective cation channel. Evaluation of PTX action suggests that the fundamental difference between channels and pumps resides not so much in the molecular architecture of the ion translocation pathway itself, by rather in the intrinsic gating properties of the protein. More importantly, the fact that high affinity toxins can induce a channel-mode of pump operation suggests that endogenous mechanisms may also exist that lead to the same channel mode and which may have either an important physiological role in cell signaling, or produce disastrous consequences for cell function and survival if not adequately controlled. Our preliminary studies have shown that another marine toxin called maitotoxin (MTX), converts the plasmalemmal Ca2+-ATPase pump (PMCA) into a Ca2+-permeable, nonselective cation channel which ultimately causes Ca2+-overload induced necrotic cell death. Furthermore, we discovered that the Ca2+ channels activated by MTX in vascular endothelial cells are biophysically identical to the channels activated by the model oxidant, tert-butyl-hydroperoxide or by oxidized glutathione (GSSG). Thus, changes in cellular redox status, appears to trigger conversion of the PMCA pump into a channel. Furthermore, our recent studies showed that the PMCA can be directly glutathionylated both in vitro and in vivo, in response to oxidant stress. Therefore, in Specific Aim #1, we will test the hypothesis that oxidative stress converts the PMCA pump into a non-selective cation channel, and in Specific Aim #2, we will determine the role of glutathionylation in both inhibition of PMCA catalytic activity and in the pump-to-channel conversion. The conversion of the PMCA pump into a channel provides a novel and ubiquitous mechanism by which oxidative stress initiates Ca2+-overload and provides a new molecular target for therapeutic intervention in a variety of pathological conditions including atherosclerosis, ischemia-reperfusion injury, Alzheimer’s disease, and biological aging.

离子通道利用存储在离子梯度中的能量来启动快速信号传导事件对 几乎体内每个细胞的功能。尽管可以响应各种频道激活 不同的刺激，一旦打开所有通道都有一个共同的特征 - 它们允许每秒数百万个离子到 越过膜。另一方面，泵送蛋白质，使用ATP中的能量来建立离子梯度 通道功能所需的。泵通常每秒移动数百个离子，因此它们的密度 在膜中，必要的要比通道高得多。这些引人注目的差异导致了视野 泵和通道通过非常不同的机制将离子跨机理移动。但是，最近 对潜在的海洋毒素Palytoxin（PTX）的研究挑战了这一概念。 PTX与皮摩尔亲和力结合 到Na+，K+-ATPase（NKA），并将泵转换为非选择性阳离子通道。 PTX的评估 行动表明，渠道和泵居住宅之间的基本差异并不多 离子易位途径本身的分子结构，而不是在固有的门控特性 蛋白质。更重要的是，高亲和力毒素可以诱导泵操作的通道模式这一事实 表明内源性机制也可能存在导致相同渠道模式的，并且可能具有 要么在细胞信号传导中起重要的身体作用，要么对细胞功能产生灾难后果 如果没有充分控制，生存也是如此。我们的初步研究表明另一种海洋毒素 称为Maitotoxin（MTX），将浆膜Ca2+-ATPase Pump（PMCA）转换为Ca2+可渗透性，非选择性 阳离子通道最终导致Ca2+超负荷引起的坏死细胞死亡。此外，我们 发现血管内皮细胞中MTX激活的Ca2+通道在生物物理上与 由模型氧化剂，TERT叔丁基 - 氧化物或氧化谷胱甘肽（GSSG）激活的通道。 那是细胞氧化还原状态的变化，似乎触发了PMCA泵转换为通道。 此外，我们最近的研究表明，PMCA可以直接在体外和IN中直接化谷胱甘肽 体内，响应氧化应激。因此，在特定的目标＃1中，我们将测试氧化的假设 压力将PMCA泵转换为非选择性阳离子通道，在特定的目标＃2中，我们将确定 谷胱甘肽化在抑制PMCA催化活性和泵到通道中的作用 转换。 PMCA泵向通道的转化提供了一种新颖而普遍的机制 氧化应力引发Ca2+​​ - 超负荷，并提供了一个新的分子靶标 干预各种病理状况，包括动脉粥样硬化，缺血 - 灌注损伤， 阿尔茨海默氏病和生物衰老。