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，这是电信号传导和二次耦合运输的基础，并且是地高辛的受体，地高辛是一种广泛使用的强心类固醇，专门抑制 Na/K泵。遗传性Na/K泵突变现在与快速发作的肌张力障碍-帕金森症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门是什么样的，以及它们精心策划的相互作用如何在细胞膜上沿相反方向传输第一个 3 个 Na，然后是 2 个 K 离子。我们将 Na/K 泵视为具有细胞质和细胞外门的特殊离子通道，其运动紧密耦合，因此两个门永远不会同时打开（与离子通道的门不同）。因此，我们通过应用强大的电生理学方法来研究 Na/K 泵，这些方法已被证明可以成功地了解离子通道的工作原理。因此，Na 和 K 离子不均匀传输产生的固定电流可以灵敏地测定稳定循环期间的周转率，电压跳跃后的电荷弛豫则可以监测限制某些部分反应速率的构象转变。这些信号共同揭示了 Na/K 泵离子传输的分子机制。现在，为了进一步研究离子传输机制，我们将这些记录方法与三种新工具结合起来：（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定点诱变，以及在非洲爪蟾卵母细胞中表达的哇巴因抗性突变体非洲爪蟾Na/K泵，其中一些带有新的半胱氨酸残基，引入该残基以测试它们对小巯基特异性试剂的可及性； （c）海洋毒素，海藻毒素，它破坏Na/K泵的两个门之间的耦合，将其转变为由泵的生理配体、Na和K离子以及核苷酸门控的离子通道。具体目标是使用这些工具：(1) 确定传输的 Na 和 K 离子穿过的离子易位途径（或多个途径）的位置、结构和物理化学特征，(2) 确定位置Na/K 泵的两个主要闸门的结构和结构，以及 (3) 检查控制闸门打开和关闭的机制。