Charge translocation by the sodium-potassium pump in the giant axon of the Humbol

描述(由申请人提供)：钠钾(Na+/K+)泵是一种重要的膜蛋白，它利用ATP的能量来运输Na+和K+，以对抗它们的电化学梯度，以维持细胞的动态平衡。本研究旨在从分子水平上理解Na~+/K~+泵输运循环中的离子事件。具体地说，我们的目标是描述和量化Na+和K+进出溶液到泵蛋白内结合位置的运动。这些实验包括使用快速电压钳在鱿鱼巨型轴突中测量电压扰动引起的电荷运动。这些实验允许测量从泵释放的三个Na+离子进入外部溶液的每个移动的时间进程。这项提议将在智利蒙特马尔的细胞生理学实验室进行，那里有来自大乌贼的1毫米轴突。有了这些轴突，电荷运动的信噪比比洛戈·佩莱伊轴突高得多，这将允许进行不可能用洛戈轴突进行的测量。其具体目的是：1)了解细胞外Na+转运转变的热力学。我们的目标是研究Na+离子在泵的外部通道内和外通道内的结合和释放过程中发生的熵和热焓变化。这将通过测量与Na+转移相关的电荷运动的所有三个分量的温度依赖性来完成。2)了解Na+/K+泵K+转运机制的生物物理性质。这一特定的目标需要全面研究K+的迁移与外界K+浓度、电压、细胞内ATP和无机磷酸盐之间的关系。这些研究有望给出由Na+/K+泵介导的电事件的机制和分子描述，Na+/K+泵是一种普遍存在的膜蛋白，在健康和疾病中具有重要意义。这项研究将主要在智利蒙特马尔与瓦尔帕莱索大学教授拉蒙·拉托雷合作进行，作为NIH第R37GM30376-29号补助金的延伸。与公共卫生相关：这里提出的实验有望直接测量Na+/K+泵内和外的离子运动，并提供泵运行期间离子结合和解离的生物物理机制。Na+/K+泵的活性对细胞内稳态至关重要，因为它有助于维持Na+和K+的梯度。这在可兴奋细胞中尤其重要，因为在动作电位过程中，负载会发生变化。临床上，Na+/K+泵很重要，因为它是地高辛、洋地黄和其他强心类固醇的受体。

DESCRIPTION (provided by applicant): The sodium potassium (Na+/K+) pump is an important membrane protein that uses the energy of ATP to transport Na+ and K+ against their electrochemical gradients to maintain cell homeostasis. This study is aimed at the molecular understanding of the ionic events in the Na+/K+ pump transport cycle. Specifically, we aim at the description and quantification of the movements of Na+ and K+ in and out from the solution to the binding sites located within the pump protein. The experiments consist of measuring the charge movement that results from voltage perturbations using a fast voltage clamp in the squid giant axon. These experiments allow the measurement of the time course of the movement of each of the three Na+ ions released from the pump into the external solution. This proposal is to be carried out at the Laboratory of Cellular Physiology in Montemar, Chile, where 1 mm axons from the squid Dosidicus gigas are available. With these axons, the signal-to-noise ratio of the charge movement is much higher than in the Loligo pealeii axons, which will allow measurements that are not feasible with Loligo axons. The specific aims are 1) understand the thermodynamics of extracellular Na+ translocation transitions. We aim at the study of the entropic and enthalpic changes that occur during the binding and release of the Na+ ions in and out of the external access channel of the pump. This will be done by measuring the temperature dependence of all three components of the charge movement associated with the Na+ translocation. 2) Understand the biophysical properties of K+ translocation mechanism of the Na+/K+ pump. This specific aim requires a comprehensive study of transitions involving the movement of K+ as a function of external concentration of K+, voltage, intracellular ATP and inorganic phospate. These studies are expected to give a mechanistic and molecular description of the electrical events mediated by the Na+/K+ pump, which is a ubiquitous membrane protein of fundamental importance in health and disease. This research will be done primarily in Montemar, Chile in collaboration with Ramon Latorre, Professor, Universidad de Valparaiso, as an extension of NIH grant No R37GM30376-29. PUBLIC HEALTH RELEVANCE: The experiments proposed here are expected to give direct measurements of the ion movements in and out of the Na+/K+ pump and provide the biophysical mechanisms of the ion binding and unbinding during the pump operation. The activity of the Na+/K+ pump is vital for cell homeostasis because it helps maintaining the Na+ and K+ gradients. It is especially important in excitable cells where the loads change during the action potential. Clinically, the Na+/K+ pump is important because it is the receptor of digoxin, digitoxin and other cardiotonic steroids.