Role for Na,K-ATPase in Nucleoplasmic Calcium Homeostasis

Na,K-ATP 酶在核质钙稳态中的作用

• 批准号: 7674984
• 负责人: CRAIG GATTO
• 金额: $ 19.18万
• 依托单位: ILLINOIS STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-17 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7674984
• 关键词: Abbreviations; Address; Amino Acids; Binding; Biochemical; Biochemistry; Calcium; Cardiac; Cardiac Glycosides; Cell Line; Cell Nucleus; Cell Surface Receptors; Cell membrane; Cell physiology; Cells; Confocal Microscopy; Congestive Heart Failure; Coupled; Cytoplasm; Data; Digitalis preparation; Drug or chemical Tissue Distribution; Electrolyte Balance; Electrophysiology (science); Endoplasmic Reticulum; Enzymes; Epileptogenesis; Epithelial Cells; Etiology; Eukaryota; Excision; Extracellular Space; Familial Hemiplegic Migraine; Foundations; Generations; Glucose; Goals; Golgi Apparatus; Health Status; Homeostasis; Hypertension; ITPR1 gene; Impaired Renal Function; Insecta; Integral Membrane Protein; Intestines; Intracellular Membranes; Investigation; Ions; Kidney; Knowledge; Laboratories; Liquid substance; Location; Maintenance; Mediating; Membrane; Molecular; Molecular Biology; Muscle; Na(+)-K(+)-Exchanging ATPase; Nature; Nerve; Nuclear; Nuclear Envelope; Nuclear Inner Membrane; Nuclear Protein; Nuclear Proteins; Nucleoplasm; Nutrient; Ouabain; Patients; Physiological; Physiology; Play; Polycystic Kidney Diseases; Protein Isoforms; Proteins; Regulation; Reporting; Research; Role; Signal Transduction; Site; Sodium; System; T-Lymphocyte; TAC1 gene; Time; Tissues; Transmembrane Transport; Work; basolateral membrane; clinically relevant; high reward; high risk; improved; inhibitor/antagonist; novel; public health relevance; receptor; research study; solute; uptake

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.

描述（由申请人提供）：钠钾三磷酸腺苷酶（即Na,K-ATP酶）负责控制高等真核生物中的细胞液体和电解质平衡。 Na,K-ATP 酶是一种异二聚体整合膜蛋白，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，这种单一酶负责利用近 40% 的细胞能量。在肾脏和肠上皮细胞中，Na,K-ATP 酶被严格递送至基底外侧膜，从而定向摄取 Na 和其他溶质（例如葡萄糖、氨基酸）。最近，一些实验室报告说，除了溶质转运之外，Na,K-ATP 酶还是强心苷介导的 Src 信号传导的细胞表面受体。 Na,K-ATP 酶的一些替代生理作用要求将酶递送到特定的亚质膜库，这提出了与 Na,K-ATP 成熟和膜靶向有关的尚未解决的问题。本提案中计划的实验将重点关注 Na,K-ATP 酶的细胞生理作用，重点关注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在内质网内组装后就可以完全发挥作用。然而，这种细胞内活动的潜在生理作用仍然是个谜。现在，最近出现的证据表明 Na/Ca 交换器在某些细胞的核钙稳态中发挥作用，这立即表明核膜上必须存在钠浓度梯度来驱动这种次级主动转运蛋白。在此应用中，我们重点证明这种细胞内 [Na+] 梯度是由在细胞内发挥作用的 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 酶驻留在 HEK-293 细胞的核膜内，并且在该膜中具有催化活性。 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，并为细胞内膜转运调节核质 Ca2+ 信号传导的新研究途径奠定基础。这些目标是：1) 证明 Na,K-ATP 酶参与核 Na+ 和 Ca2+ 转运，2) 鉴定将 Na,K-ATP 酶保留在核膜内的蛋白质，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、电生理学和共焦显微镜的策略来实现我们的科学目标。 Na,K-ATP酶是强心苷的药理学靶点，强心苷是一种广泛用于治疗充血性心力衰竭的药物。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，本文提出的工作对于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、家族性偏瘫性偏头痛、癫痫发生和多囊肾病。公众健康相关性：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，改进心脏功能治疗以及改善受损肾功能的前景将得到极大帮助。