Cell Structure-Function of Na pump Assembly

描述（由申请人提供）：Na，K-ATPase负责控制高等真核生物的细胞液和电解质平衡。它是一种异二聚体整合膜蛋白，由一个催化亚基（~110 kDa）和一个糖基化2亚基（~55 kDa）组成。在某些生理状态下，这种单一的酶负责利用近40%的细胞能量。在肾脏和肠上皮细胞中，Na，K-ATP酶严格地递送至基底外侧膜，提供Na+和其他溶质（例如葡萄糖、氨基酸）的定向摄取。最近，一些实验室报道，除了溶质转运，Na，K-ATP酶是内源性心脏糖苷介导的Src信号传导的细胞表面受体。Na，K-ATP酶的这些替代生理作用中的一些要求将酶递送至特定的亚质膜池，这提出了与Na，K-ATP酶成熟和运输有关的尚未解决的问题。本提案中计划的实验将侧重于Na，K-ATP酶的成熟和组装，重点是寡聚化在膜递送中的作用。我们的初步数据已经导致了总体假设：Na，K-ATP酶寡聚化对于适当的膜递送是至关重要的。我们将在拟议的研究中验证这一假设。本提案中概述的具体实验将利用昆虫细胞表达，缺乏内源性Na，K-ATP酶，以解决未解决的问题，组装，运输，和寡聚状态的Na泵途中质膜。通过杆状病毒系统将野生型和突变型绵羊a-亚基引入昆虫细胞中，并通过膜分离和免疫共沉淀从内质网到质膜进行泵成熟过程。在目标3中，我们将确认并将我们在昆虫细胞中的体外观察扩展到哺乳动物细胞（即HEK-293细胞）中的原位测量。本工作的结果将提供有关Na，K-ATP酶四级结构及其从内质网出口到质膜的重要性的重要新信息。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，以实现我们的科学目标。Na，K-ATP酶是强心苷的药理学靶点，强心苷是充血性心力衰竭的广泛治疗方法。考虑到越来越多的证据表明，Na，K-ATP酶的细胞分布在其生理学中起着关键作用，这里提出的工作将是至关重要的理解和解决临床相关问题的病因。具体而言，Na，K-ATP酶的失调归因于高血压、充血性心力衰竭、家族性偏瘫性偏头痛和多囊肾病。 公共卫生相关性：Na，K-ATP酶是一种重要的转运系统，也是洋地黄的作用部位，洋地黄是治疗充血性心力衰竭患者最广泛使用的疗法。当我们更好地理解细胞膜中Na，K-ATP酶活性的调节，并阐明细胞将这种重要酶适当递送到特定亚细胞位置的机制时，将极大地有助于改善心脏功能的治疗前景，以及改善受损的肾功能。

DESCRIPTION (provided by applicant): The Na,K-ATPase is responsible for controlling cellular fluid and electrolyte balance in higher eukaryotes. It is a heterodimeric integral membrane protein consisting of a catalytic ¿-subunit (~110 kDa) and a glycosylated 2-subunit (~55 kDa). During some physiological states, this single enzyme is responsible for utilizing nearly 40% of the cells energy. In kidney and intestinal epithelial cells the Na,K-ATPase is strictly delivered to the basolateral membrane providing directional uptake of Na+ and other solutes (e.g. glucose, amino acids). Recently, several laboratories have reported that in addition to solute transport, the Na,K-ATPase is a cell-surface receptor for endogenous cardiac glycoside-mediated Src signaling. Some of these alternative physiological roles of the Na,K-ATPase mandate that the enzyme be delivered to specific sub-plasma membrane pools, which raises as yet unaddressed questions pertaining to Na,K- ATPase maturation and trafficking. The planned experiments in this proposal will focus on the maturation and assembly of Na,K-ATPase, focusing on the role that oligomerization plays in membrane delivery. Our preliminary data has led to the overall hypothesis: Na,K-ATPase oligomerization is critical for proper membrane delivery. We will test this hypothesis in the proposed studies. The specific experiments outlined in this proposal will exploit insect cell expression, devoid of endogenous Na,K-ATPase, to address unresolved issues concerning the assembly, trafficking, and oligomeric state of the Na pump en route to the plasma membrane. Wild-type and mutant sheep a-subunits will be introduced into the insect cells via the baculovirus system and the pump maturation process will be followed from endoplasmic reticulum to plasma membrane by membrane fractionation and co-immunoprecipitation. In Aim 3, we will confirm and extend our in vitro observations in insect cells to in situ measurements in mammalian cells (i.e. HEK-293 cells). The results from this work will provide important new information about Na,K-ATPase quaternary structure and its importance for exit from the endoplasmic reticulum and delivery to the plasma membrane. We will combine strategies from molecular biology, cell physiology, biochemistry and confocal microscopy to accomplish our scientific goals. The Na,K-ATPase is the pharmacological target for cardiac glycosides, a widely used therapy for congestive heart failure. Considering the mounting evidence that cellular distribution of Na,K-ATPase plays a key role in its physiology, the work proposed here will be crucial to understanding and resolving the etiology of clinically relevant problems. Specifically, dysregulation of the Na,K-ATPase has been attributed to hypertension, congestive heart failure, familial hemiplegic migraine and polycystic kidney disease. PUBLIC HEALTH RELEVANCE: The Na,K-ATPase is an essential transport system and the site of action of digitalis, the most widely used therapy to treat patients with congestive heart failure. Prospects for improved therapies for cardiac function, as well as improving impaired renal function, will be greatly aided when we have a better understanding of the regulation of the activity of the Na,K-ATPase in cell membranes and by elucidating the mechanisms by which cells properly deliver this vital enzyme to specific subcellular locations.