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的能量运输Na+和K+对抗其电化学梯度以维持细胞稳态。本研究旨在对Na+/K+泵输运循环中的离子事件进行分子理解。具体来说，我们的目标是描述和量化Na+和K+进出溶液到位于泵蛋白内的结合位点的运动。实验包括在鱿鱼巨型轴突中使用快速电压钳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到外部溶液中的三个Na+离子的运动的时间过程。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1毫米长的鱿鱼轴突。在这些轴突中，电荷运动的信噪比比在Loligo pealeii轴突中要高得多，这将允许在Loligo轴突中无法实现的测量。具体目标是1)了解细胞外Na+易位转变的热力学。我们的目的是研究Na+离子在泵的外部通道的结合和释放过程中发生的熵和焓的变化。这将通过测量与Na+易位相关的电荷运动的所有三个组成部分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。为了实现这一特定目标，需要对K+的运动作为外部K+浓度、电压、细胞内ATP和无机磷酸盐的函数进行全面研究。这些研究有望对Na+/K+泵介导的电事件进行机制和分子描述，Na+/K+泵是一种普遍存在的膜蛋白，对健康和疾病具有重要意义。这项研究将主要在智利蒙特马尔进行，与瓦尔帕莱索大学教授Ramon Latorre合作，作为NIH拨款No . R37GM30376-29的延伸。公共卫生相关性：这里提出的实验有望直接测量进出Na+/K+泵的离子运动，并提供泵运行期间离子结合和解结合的生物物理机制。Na+/K+泵的活性对细胞稳态至关重要，因为它有助于维持Na+和K+梯度。在动作电位期间负荷变化的可兴奋细胞中，这一点尤为重要。临床上，钠离子/钾离子泵很重要，因为它是地高辛、地黄霉素和其他强心剂类固醇的受体。

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.