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泵生成了基于电信号传导和二次耦合传输的Na和K离子的跨膜电化学梯度，并且是高氧蛋白的受体，高氧蛋白是广泛规定的心脏固醇类固醇，专门抑制Na/k泵。遗传的Na/k泵突变与快速发作的肌张力障碍 - - 帕金森氏症和家族性偏瘫偏头痛有关。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大门是什么样的，以及它们的策划交互如何在整个细胞膜的方向上以相反的方向运输前3 Na，然后将2 K离子运输。我们将Na/k泵视为具有细胞质和细胞外门的专业离子通道，其运动紧密耦合，因此两个门通常都不会同时打开（与离子通道的门不同）。因此，我们通过应用强大的电生理方法来研究Na/k泵，这些方法已证明成功地学习了离子通道的工作方式。因此，由Na和K离子的不平等运输导致的固定电流在稳定循环过程中敏感地测定周转率，并且电压跳跃后的电荷松弛监测构象转变，以限制某些部分部分反应。这些信号共同阐明了Na/K泵的离子转运的分子机理。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相结合：（a）基于最近的高分辨率晶体结构的Na/k泵α亚基的同源模型，相关SR CA泵的关键构型； （b）基于这些结构模型的位点特异性诱变，以及在异武牛卵母细胞中的表达，对ouaabain耐药突变体xenopus na/k泵的表达，一些带有新型的半胱氨酸残基，以测试其对小硫化硫二酰基特异性试剂的可及性； （c）海洋毒素，palytoxin，它破坏了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离子和核苷酸的离子通道。具体目的是使用这些工具：（1）确定由运输的Na和k离子穿过的离子转换途径（或途径）的位置，结构和物理化学特征（2）确定Na/K泵的位置和结构，以确定NA/K泵的两个主要门口的位置和结构，以及（3），以及（3），以及（3），以检验开放和封闭的机构。