The Roles of Sodium Pump Beta Subunits in Mammalian Cells

描述（由申请人提供）：钠泵负责调节和控制细胞、器官和整个生物体中的液体和电解质平衡。它是一种完整的膜蛋白，利用细胞ATP水解的能量，利用细胞能量的40%主动转运Na和K离子，对抗它们的梯度。该泵是由两个主要的子单元，？亚基，约1000个氨基酸的催化亚基和一个？约300个氨基酸残基的亚基，其被高度糖基化。钠泵，或Na，K-ATP酶，是盐和液体调节所必需的，并且仅在肾脏和肠的极化上皮细胞的基底外侧膜中发现。计划中的实验将集中在钠泵的细胞生理作用，重点是？？哺乳动物细胞中的亚基。我们以前的工作导致了一个假设如何？亚基促进钠泵释放？？复合物从内质网中释放出来，并使其能够传递到质膜。我们计划在拟议的研究中验证这一假设。我们还将研究钠泵所发挥的作用？亚基在上皮细胞中建立和维持细胞-细胞接触。我们已经发现了钠泵的唯一基底外侧递送可以被超越并且其被递送到顶面的情况。我们计划利用这些协议，以了解如何控制极化膜交付。在这些实验中，我们还将探讨的生理作用？亚基糖基化，这是目前未知的。我们已经开发了一种调节表达系统，用于增加？哺乳动物细胞中的亚基和用于其减少的基于RNA的方法。我们将结合联合收割机的策略，从分子生物学，细胞生理学，生物化学和共聚焦显微镜，以追求我们的科学目标。细胞的作用？钠泵的亚基还没有很好的理解。钠泵是参与液体和电解质平衡的主要膜蛋白，并且是强心苷类（例如洋地黄）的主要靶点，洋地黄是用于心功能不全的最广泛的疗法。钠泵的失调也与几种疾病状态有关，包括高血压和偏头痛。本申请中提出的工作将提供有关这种重要蛋白质如何在细胞中发挥其功能的新信息。

DESCRIPTION (provided by applicant): The sodium pump is responsible for the regulation and control of fluid and electrolyte balance in cells, organs and in the entire organism. It is an integral membrane protein that utilizes as much as 40% of the energy of cells to actively transport Na and K ions against their gradients utilizing the energy of hydrolysis of cellular ATP. The pump is composed of two main subunits, the ? subunit, the catalytic subunit of about 1000 amino- acids and a ? subunit of about 300 amino-acid residues which is heavily glycosylated. The sodium pump, or Na,K-ATPase, is essential for salt and fluid regulation and is found exclusively in the basolateral membrane of polarized epithelial cells of the kidney and intestines. The planned experiments will focus on the cell physiological roles of the sodium pump, focusing on the multiple roles of the ??subunit in mammalian cells. Our previous work has led to a hypothesis of how the ? subunit facilitates release of the Na pump ?? complex from the endoplasmic reticulum and enables its delivery to the plasma membrane. We plan to test this hypothesis in the proposed studies. We will also examine the role played by the Na pump ? subunit in establishing and maintaining cell-cell contacts in epithelial cells. We have discovered conditions where the exclusive basolateral delivery of the sodium pump can be over-ridden and it is delivered to the apical surface. We plan to utilize these protocols to understand how the polarized membrane delivery is controlled. Throughout these experiments we will also probe the physiological role of ? subunit glycosylation, which is currently unknown. We have developed a regulated expression system for increasing the levels of the ? subunit in mammalian cells and RNA-based methods for its reduction. We will combine strategies from molecular biology, cell physiology, biochemistry and confocal microscopy to pursue our scientific goals. The cellular roles of the ? subunit of the sodium pump are not well understood. The sodium pump is the primary membrane protein involved in fluid and electrolyte balance and is the major target of cardiac glycosides, such as digitalis, the most widely used therapy for cardiac insufficiency. Disregulation of the sodium pump is also associated with several disease states, including hypertension and migraines. The work proposed in the current application will provide new information about how this important protein carries out its functions in cells.