STRUCTURE/FUNCTION OF THE NA/K PUMP

NA/K 泵的结构/功能

• 批准号: 6351872
• 负责人: MIGUEL HOLMGREN
• 金额: $ 26.37万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-02-01 至 2001-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6351872
• 关键词: animal genetic material tag; biophysics; cysteine; electrical measurement; electrophysiology; enzyme mechanism; enzyme structure; hydropathy; intracellular transport; ion transport; membrane channels; ouabain; potassium ion; protein structure function; site directed mutagenesis; sodium ion; sodium potassium exchanging ATPase; sulfhydryl reagents; tissue /cell culture; transfection; transfection /expression vector

The long term objective of this project is an understanding, at a molecular level, of how the Na/K pump works. This virtually ubiquitous enzyme uses the energy from the hydrolysis of ATP to transport Na+ and K+ ions against their electrochemical gradient. By doing so, the Na/K pump maintains the K gradient that generates the transmembrane resting potential and the Na gradient which is used to produce electrical signals as well as to drive the uphill transport across the cell membrane of other substances. These ionic gradients are also responsible for cell volume regulation. Clinically, the Na/K pump is important because it is the receptor of digoxin, a widely prescribed cardiac steroid used to control some cardiac arrhythmias. In each pump cycle, three Na+ ions and two K+ ions are translocated through the protein. When ions are moved through the protein, electrical charge is translocated which can be measured and used to learn some properties of the movement of ions through the protein. To achieve the objective, voltage-induced steady and transient currents elicited by Na/K pumps will be measured in two preparations: the cloned rat brain pump expressed in a mammalian cell line and the endogenous pump from squid giant axons. For the recombinant rat brain pump, the possibility to manipulate the protein itself will be used to learn about the structure and function of the Na/K pump. Site-directed mutagenesis of selected residues to cysteine, and subsequent chemical modification, will be used to map the exposed surface of the protein. We will also exploit the interaction of palytoxin with the Na/K pump. Palytoxin is a marine toxin that is believed to transform the pump into a "channel", so that ions then move at rates 10,000 times faster than through the normal pumps. In the squid giant axon preparation, the access to internal and external solutions as well as the very fast voltage-clamp will be exploited to learn about the mechanism of ion translocation, particularly for Na+ ions. The three specific aims for this project are (i) to understand the ion-channel-like behavior by the Na/K pump, (ii) to determine the exposed surface of the pathways used by Na+ and K+ ions as they move through the protein, and (iii) to pursue the biophysical properties of the Na+ ion translocation mechanism through the Na/K pump.

该项目的长期目标是一种理解， Na/k泵的工作方式的分子水平。 这实际上无处不在 酶利用来自ATP水解到运输Na+的能量 K+离子反对其电化学梯度。这样，Na/k 泵保持生成跨膜静止的K梯度 电势和用于产生电气的NA梯度 信号以及驾驶越过牢房的上坡运输 其他物质的膜。 这些离子梯度也是 负责细胞体积调节。 临床上，Na/K泵为 重要的是因为它是地高辛的受体，一种广泛的处方 心脏类固醇用于控制某些心律不齐。 在每个泵中 循环，三个Na+离子和两个K+离子通过 蛋白质。 当离子通过蛋白质移动时，电荷为 易位，可以测量并用于学习的某些特性 离子通过蛋白质的运动。 为了实现目标， 电压引起的稳定和瞬态电流由Na/K泵引起 将在两次制剂中测量：克隆的大鼠脑泵 在哺乳动物细胞系和鱿鱼的内源性泵中表达 巨型轴突。 对于重组大鼠脑泵， 操纵蛋白质本身将用于学习结构 Na/K泵的功能。 选定的位置定向诱变 将使用半胱氨酸的残留物以及随后的化学修饰 绘制蛋白质的暴露表面。 我们还将利用 呼毒素与Na/k泵的相互作用。 palytoxin是一种海洋毒素 据信这将泵转换为“频道”，以便离子 然后以比普通泵快10,000倍的速度移动。 在鱿鱼巨型轴突准备中，进入内部和外部 解决方案以及非常快速的电压夹将被利用为 了解离子易位的机制，特别是对于Na+ 离子。 该项目的三个具体目标是（i）了解 Na/K泵的离子通道样行为（ii）以确定 Na+和K+离子移动时使用的途径的表面 通过蛋白质和（iii）追求生物物理特性 Na+离子易位机制通过Na/K泵。