Role for Na,K-ATPase in Nucleoplasmic Calcium Homeostasis

描述（由申请人提供）：钠钾腺苷三磷酸酶（即Na， k - atp酶）负责控制高等真核生物的细胞液和电解质平衡。Na， k - atp酶是一种异二聚体整体膜蛋白，由催化a-亚基（约1000个氨基酸）和糖基化¿-亚基（约300个氨基酸）组成。在某些生理状态下，这种酶负责利用近40%的细胞能量。在肾和肠上皮细胞中，Na， k - atp酶被严格地传递到基底外侧膜，提供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，Na， k - atp酶是心脏糖苷介导Src信号传导的细胞表面受体。这些Na，K-ATP酶的一些替代生理作用要求酶被递送到特定的亚质膜池，这就提出了关于Na，K-ATP成熟和膜靶向的尚未解决的问题。本课题计划的实验将集中于Na， k - atp酶的细胞生理作用，重点研究一种新的细胞内生理功能。我们之前的工作首次揭示了钠泵一旦在内质网内组装就完全起作用。然而，这种细胞内活动的潜在生理作用仍然是一个谜。最近有证据表明，钠钙交换器在某些细胞的核钙稳态中起作用，这立即表明，在核膜上必须存在钠浓度梯度来驱动这种二级活性转运体。在本应用中，我们着重于证明这种细胞内[Na+]梯度是由细胞内功能的Na， k - atp酶建立和维持的。事实上，我们提供的初步数据表明，Na， k - atp酶存在于HEK-293细胞的核膜内，并在该膜上具有催化活性。在R21提案中提出的具体目标将提供有关Na， k - atp酶的重要新信息，并为研究细胞膜内转运调节核质Ca2+信号传导的新途径提供基础。这些目标是：1)证明Na， k - atp酶参与核Na+和Ca2+运输，2)鉴定核膜内保留Na， k - atp酶的蛋白质，以及3)确定核Na， k - atp酶靶向的组织分布。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完成我们的科学目标。Na， k - atp酶是心脏糖苷的药理学靶点，广泛用于治疗充血性心力衰竭。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，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。具体来说，Na， k - atp酶的失调可归因于高血压、充血性心力衰竭、家族性偏瘫偏头痛、癫痫发生和多囊肾病。公共卫生相关性：Na， k - atp酶是重要的转运系统和洋地黄的作用部位，洋地黄是治疗充血性心力衰竭患者最广泛使用的药物。当我们更好地了解细胞膜中Na， k - atp酶活性的调节，并阐明细胞正确地将这种重要酶传递到特定亚细胞位置的机制时，将大大有助于改善心功能和改善肾功能受损的治疗方法的前景。

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.