Regulation of PMCA Pump-Channels by Oxidant Stress

离子通道使用储存在离子梯度中的能量来启动对细胞的增殖至关重要的快速信号传导事件。 身体中几乎每个细胞的功能。尽管通道激活可以响应于多种信号而发生， 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， 穿过膜。另一方面，泵蛋白利用ATP中的能量建立离子梯度 渠道功能的必要性。泵通常每秒移动数百个离子，因此它们的密度 在膜中的浓度必然比通道中的浓度高得多。这些显著的差异导致了这样一种观点， 泵和通道通过非常不同的机制使离子穿过膜。但最近的 对有效的海洋毒素-海葵毒素（PTX）的研究挑战了这一概念。PTX以皮摩尔亲和力结合 与Na+，K+-ATP酶（NKA）连接，并将泵转化为非选择性阳离子通道。PTX评价 行动表明，渠道和泵之间的根本区别并不在于 离子易位途径本身的分子结构，而不是在内在的门控性质的离子转运途径。 蛋白更重要的是，事实上，高亲和力毒素可以诱导泵操作的通道模式， 表明，内源性机制也可能存在，导致相同的通道模式，并可能有 要么在细胞信号传导中发挥重要的生理作用，要么对细胞功能产生灾难性的后果 如果不加以充分控制，我们的初步研究表明另一种海洋毒素 称为maitotoxin（MTX），将质膜Ca 2 +-ATP酶泵（PMCA）转化为Ca 2+渗透性，非选择性 阳离子通道，最终导致Ca 2+超载诱导的坏死细胞死亡。而且我们 发现在血管内皮细胞中由MTX激活的Ca 2+通道与 由模型氧化剂、叔丁基氢过氧化物或氧化型谷胱甘肽（GSSG）激活的通道。 因此，细胞氧化还原状态的变化似乎触发PMCA泵转化为通道。 此外，我们最近的研究表明，PMCA在体外和体内都可以直接谷胱甘肽化， 体内，对氧化应激的反应。因此，在具体目标#1中，我们将检验氧化性 压力将PMCA泵转化为非选择性阳离子通道，在具体目标#2中，我们将确定 谷胱甘肽化在抑制PMCA催化活性和泵-通道中的作用 转换.将PMCA泵转换为通道提供了一种新颖且普遍存在的机制 通过氧化应激引发Ca 2+超载，并为治疗提供新的分子靶点， 干预多种病理状况，包括动脉粥样硬化，缺血-再灌注损伤， 老年痴呆症和生物衰老。

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.