MOLECULAR PHYSIOLOGY OF THE SODIUM PUMP

描述(由申请人提供)：本工作的目标是对Na泵或Na，K-ATPase的分子生理学有更全面的了解。钠泵将Na和K离子在几乎所有哺乳动物细胞的质膜上的主动运输与ATP水解酶的催化作用结合在一起。钠泵负责维持正常的液体和电解质平衡，对肾脏功能、能量再吸收和营养吸收至关重要。在过去的几十年里，我们已经了解了很多关于这种寡聚蛋白如何执行其重要功能的知识。然而，对亚基在合成后组装并作为一个功能单位输送到质膜的过程的控制和调节知之甚少。在目前的工作中，我们将利用杆状病毒感染的昆虫细胞系统将钠泵亚单位引入正常不表达它们的细胞中。我们将分别和一起检测绵羊α1亚基(1000个氨基酸的多聚体膜蛋白)和绵羊β1亚基(约300个氨基酸的单膜交叉蛋白)的表达，并利用细胞分离和代谢标记策略研究控制它们在内质网中组装和通过高尔基体传递到质膜的因素。通过利用每个亚单位中的一系列突变，我们将研究影响向质膜输送的分子决定因素的作用，它在膜上的稳定性以及它与细胞骨架元素的相互作用。钠泵是一种重要的运输系统，也是洋地黄的作用部位，洋地黄是治疗心功能不全最广泛使用的疗法。当我们更好地了解细胞膜上钠泵活动的调节时，改善心脏功能和改善肾功能受损的治疗方法的前景将大大有助于我们。

DESCRIPTION (provided by applicant): The objective of the present work is to obtain a more complete understanding of the molecular physiology of the Na pump or Na,K-ATPase. The Na pump couples the active transport of Na and K ions across the plasma membrane of almost all mammalian cells to the catalysis of ATP hydrolysis. The Na pump is responsible for maintaining normal fluid and electrolyte balance and is essential for renal function and energising reabsorbtion and nutrient uptake. During the past several decades we have learned much about how this oligomeric protein carries out its important function. However, little is known about the control and regulation of the processes which enable the subunits to assemble after synthesis and be delivered as a functioning unit to the plasma membrane. In the present work we will employ the baculovirus-infected insect cell system to introduce Na pump subunits into cells which do not normally express them. We will examine the expression of the sheep alpha1 -subunit (a polytopic membrane protein of 1000 amino acids) and sheep Beta1-subunit (a protein of about 300 amino acids and a single membrane crossing) separately and together and using cell fractionation and metabolic labelling strategies investigate the factors which control their assembly in the endoplasmic reticulum and delivery to the plasma membrane via the golgi apparatus. By utilizing a series of mutations in each sub-unit we will investigate the role of molecular determinants which affect delivery to the plasma membrane, its stability in the membrane and its interactions with cytoskeletal elements. The Na pump is an essential transport system and the site of action of digitalis, the most widely used therapy in cardiac insufficiency. Prospects for improved therapies for cardiac function and for improving impaired renal function will be greatly aided when we have a better understanding of the regulation of the activity of the Na pump in cell membranes.