Cell Structure-Function of Na pump Assembly

描述(申请人提供)：在高等真核生物中，Na，K-ATPase负责控制细胞液和电解质平衡。它是由一个催化亚基(~110 kDa)和一个糖基化的2亚基(~55 kDa)组成的异二聚体完整膜蛋白。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。在肾和肠上皮细胞中，Na，K-ATPase被严格输送到基底膜，提供对Na+和其他溶质(如葡萄糖、氨基酸)的定向摄取。最近，一些实验室报道，除了溶质运输外，Na，K-ATPase是内源性心脏糖苷介导的Src信号的细胞表面受体。Na，K-ATPase的一些替代生理作用要求酶被输送到特定的亚质膜池，这就提出了与Na，K-ATPase成熟和运输有关的尚未解决的问题。这项计划中计划的实验将集中在Na，K-ATPase的成熟和组装上，重点放在齐聚在膜传递中所起的作用。我们的初步数据导致了总体假设：Na，K-ATPase寡聚对适当的膜输送至关重要。我们将在拟议的研究中检验这一假设。该提案中概述的具体实验将利用缺乏内源性Na，K-ATPase的昆虫细胞表达来解决尚未解决的问题，即Na泵在通往质膜的途中的组装、运输和寡聚体状态。野生型和突变型绵羊α亚基将通过杆状病毒系统导入昆虫细胞，通过膜分离和免疫共沉淀，泵的成熟过程将从内质网进入质膜。在目标3中，我们将确认和扩展我们在昆虫细胞中的体外观察，并将其扩展到哺乳动物细胞(即HEK-293细胞)的原位测量。这项工作的结果将为Na，K-ATPase的四级结构及其在离开内质网和向质膜输送方面的重要性提供重要的新信息。我们将结合分子生物学、细胞生理学、生物化学和共聚焦显微镜的策略来实现我们的科学目标。Na，K-ATPase是一种广泛用于治疗充血性心力衰竭的心苷类药物的药理靶点。考虑到越来越多的证据表明Na，K-ATPase的细胞分布在其生理学中起着关键作用，本文提出的工作将对理解和解决临床相关问题的病因至关重要。具体地说，Na，K-ATPase的失调被归因于高血压、充血性心力衰竭、家族性偏瘫、偏头痛和多囊肾病。 公共卫生相关性：Na，K-ATPase是一种基本的运输系统，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。当我们更好地了解细胞膜上Na，K-ATPase活性的调节，并通过阐明细胞将这种重要的酶适当地输送到特定的亚细胞位置的机制时，改善心脏功能的治疗以及改善受损的肾功能的前景将得到极大的帮助。

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.