Na/K Pump Current in Isolated Heart Cells

离体心脏细胞中的 Na/K 泵电流

• 批准号: 7822168
• 负责人: DAVID C GADSBY
• 金额: $ 0.67万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-06-01 至 2009-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7822168
• 关键词: Axon; Binding Sites; Biological Assay; Cardiac Glycosides; Cardiac Myocytes; Cavia; Cell membrane; Cells; Characteristics; Charge; Chemicals; Core Protein; Coupled; Coupling; Cysteine; Dystonia 12; Embryo; Familial Hemiplegic Migraine; Funding; Gated Ion Channel; Goals; Grant; Homology Modeling; Human; Inherited; Ion Channel; Ion Channel Gating; Ion Pumps; Ion Transport; Ions; Learning; Life; Ligands; Light; Link; Location; Marine Toxins; Measurement; Measures; Membrane; Membrane Potentials; Methods; Molecular; Molecular Conformation; Monitor; Movement; Muscle Cells; Mutation; Na(+)-K(+)-Exchanging ATPase; Nucleotides; Oocytes; Ouabain; Pathway interactions; Physiological; Proteins; Pump; Reaction; Reagent; Relaxation; Resistance; Resolution; Side; Signal Transduction; Site-Directed Mutagenesis; Solutions; Squid; Structural Models; Structure; System; Testing; Time; Transport Reaction; Ventricular; Work; Xenopus; Xenopus oocyte; base; conformational conversion; digoxin receptor; extracellular; heart cell; kidney cell; millisecond; mutant; novel; palytoxin; reagent testing; research study; stoichiometry; tool; voltage; voltage clamp

DESCRIPTION (provided by applicant): The Na/K pump generates the transmembrane electrochemical gradients of Na and K ions that underlie electrical signaling and secondary coupled transport, and is the receptor for digoxin, the widely prescribed cardiotonic steroid that specifically inhibits the Na/K pump. Inherited Na/K pump mutations are now linked to rapid-onset dystonia-parkinsonism and familial hemiplegic migraines. Our long term goal remains to understand in detail how the Na/K pump works, i.e. what the ion translocation pathways and associated gates look like, and how their orchestrated interaction transports first 3 Na and then 2 K ions in opposite directions across the cell membrane. We view the Na/K pump as a specialized ion channel with cytoplasmic and extracellular gates whose movements are tightly coupled so that both gates are never normally open at the same time (unlike the gates of ion channels). Accordingly, we investigate the Na/K pump by applying powerful electrophysiological methods that have proven successful in learning how ion channels work. Thus, the stationary current that results from the unequal transport of Na and K ions sensitively assays turnover rate during steady cycling, and charge relaxations following voltage jumps monitor conformational transitions that rate limit certain partial reactions. Together, these signals have shed light on the molecular mechanism of ion transport by the Na/K pump. Now, to further investigate the ion transport mechanism, we combine these recording methods with three new tools: (a) homology models of the Na/K pump alpha subunit based on recent high-resolution crystal structures of key conformations of the related SR Ca pump; (b) site-specific mutagenesis based on those structural models, and expression in Xenopus oocytes, of ouabain-resistant mutant Xenopus Na/K pumps, some bearing novel cysteine residues introduced to test their accessibility to small sulfhydryl-specific reagents; (c) the marine toxin, palytoxin, which disrupts the coupling between the Na/K pump's two gates, transforming it into an ion channel gated by the pump's physiological ligands, Na and K ions and nucleotides. The specific aims are to use these tools: (1) to determine the location, structure, and physico-chemical characteristics of the ion-translocation pathway (or pathways) traversed by the transported Na and K ions, (2) to determine the location and structure of the Na/K pump's two principal gates, and (3) to examine the mechanisms controlling opening and closing of the gates.

描述（由申请人提供）：Na/K泵产生Na和K离子的跨膜电化学梯度，这是电信号和次级偶联转运的基础，并且是地高辛的受体，地高辛是广泛处方的强心类固醇，特异性抑制Na/K泵。遗传性Na/K泵突变现在与快速发作的肌张力障碍-帕金森综合征和家族性偏瘫性偏头痛有关。我们的长期目标仍然是详细了解Na/K泵的工作原理，即离子易位途径和相关的门是什么样子的，以及它们的协调相互作用如何以相反的方向首先运输3个Na离子，然后运输2个K离子穿过细胞膜。我们将Na/K泵视为具有细胞质和细胞外门的专门离子通道，其运动紧密耦合，使得两个门通常不会同时打开（不像离子通道的门）。因此，我们通过应用强大的电生理方法来研究Na/K泵，这些方法已被证明在了解离子通道如何工作方面是成功的。因此，静态电流，导致从Na和K离子的不平等运输灵敏地测定周转率在稳定的循环，和电荷弛豫电压跳变监测构象转变，速率限制某些部分反应。总之，这些信号揭示了Na/K泵离子转运的分子机制。现在，为了进一步研究离子转运机制，我们将这些记录方法与三种新工具相结合：（a）基于相关SR Ca泵关键构象的最新高分辨率晶体结构的Na/K泵α亚基的同源模型;（B）基于这些结构模型的位点特异性诱变，以及哇巴因抗性突变体爪蟾Na/K泵在爪蟾卵母细胞中的表达，一些带有新的半胱氨酸残基，引入以测试它们对小巯基特异性试剂的可及性;（c）海洋毒素，海葵毒素，其破坏Na/K泵的两个门之间的耦合，将其转化为由泵的生理配体、Na和K离子以及核苷酸门控的离子通道。具体的目的是使用这些工具：（1）确定位置，结构和物理化学特性的离子转运途径（或途径）通过运输Na和K离子，（2）确定的位置和结构的Na/K泵的两个主要的门，和（3）检查的机制控制打开和关闭的门。