Role for Na,K-ATPase in Nucleoplasmic Calcium Homeostasis

描述(申请人提供)：钠钾三磷酸腺苷酶(即Na，K-ATPase)在高等真核生物中负责控制细胞内的液体和电解质平衡。Na，K-ATPase是由催化α亚基(~1000个氨基酸)和糖基化β亚基(~300个氨基酸)组成的异源二聚体完整膜蛋白。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是心脏糖苷介导的Src信号的细胞表面受体。其中一些Na，K-ATPase的替代生理作用要求酶被输送到特定的亚质膜池，这提出了与Na，K-ATP成熟和膜靶向有关的尚未解决的问题。计划中的实验将集中在Na，K-ATPase的细胞生理作用上，重点放在一种新的细胞内生理功能上。我们之前的工作第一次揭示了钠泵一旦组装在内质网中就可以完全发挥功能。然而，这种细胞内活动的潜在生理作用仍然是一个谜。最近出现的证据表明，Na/Ca交换器在一些细胞的核内钙稳态中发挥作用，这立即表明，必须有一个跨越核膜的钠浓度梯度来驱动这个二级活性转运蛋白。在这一应用中，我们着重于证明这种细胞内的[Na+]梯度是由细胞内的Na，K-ATPase功能建立和维持的。事实上，我们提供的初步数据表明，Na，K-ATPase位于HEK-293细胞的核膜内，并在这一膜上具有催化活性。这一R21提案中涉及的特定目标将提供关于Na，K-ATPase的重要新信息，并为研究调节核质钙信号的细胞膜运输提供新的途径。这些目的是：1)证明Na，K-ATPase参与核内Na+和Ca~(2+)的转运，2)鉴定核膜内保留Na，K-ATPase的蛋白质，3)确定核内Na，K-ATPase靶向的组织分布。我们将结合分子生物学、细胞生理学、生物化学、电生理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、癫痫的发生和多囊肾病。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 sodium-potassium adenosine triphosphatase (i.e. Na,K-ATPase) is responsible for controlling cellular fluid and electrolyte balance in higher eukaryotes. The Na,K-ATPase is a heterodimeric integral membrane protein consisting of a catalytic a-subunit (~1000 amino acids) and a glycosylated ¿-subunit (~300 amino acids). 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 cardiac glycoside-mediating 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-ATP maturation and membrane targeting. The planned experiments in this proposal will focus on the cell physiological roles of Na,K-ATPase, focusing on a novel intracellular physiological function. Our previous work revealed for the first time that the Na pump is completely functional once assembled within the endoplasmic reticulum. However, the potential physiological role of this intracellular activity remained a mystery. Now recent evidence has emerged demonstrating that the Na/Ca exchanger plays a role in nuclear calcium homeostasis in some cells which immediately suggests that there must be a sodium concentration gradient across the nuclear envelope to drive this secondary active transporter. In this application, we focus on demonstrating that this intracellular [Na+] gradient is established and maintained by the Na,K-ATPase functioning intracellularly. Indeed, we provide preliminary data showing that the Na,K-ATPase resides within the nuclear envelope in HEK-293 cells and is catalytically active in this membrane. The specific aims addressed in this R21 proposal will provide important new information about Na,K-ATPase and provide the foundation for a new avenue of investigation regarding intracellular membrane transport to regulate nucleoplasmic Ca2+ signaling. These aims are: 1) Demonstrate that the Na,K-ATPase participates in nuclear Na+ and Ca2+ transport, 2) Identify proteins that retain Na,K-ATPase within the nuclear envelope, and 3) Determine the tissue distribution of nuclear Na,K-ATPase targeting. We will combine strategies from molecular biology, cell physiology, biochemistry, electrophysiology 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, epileptogenesis, 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.