VOLTAGE CLAMP STUDIES OF SODIUM PUMP CURRENT AND FLUX

钠泵电流和通量的电压钳研究

• 批准号: 6323707
• 负责人: ROBERT F RAKOWSKI
• 金额: $ 5万
• 依托单位: ROSALIND FRANKLIN UNIV OF MEDICINE & SCI
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-05-01 至 2000-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6323707
• 关键词: Xenopus; Xenopus oocyte; adenosine triphosphate; aminoacid; animal genetic material tag; axon; enzyme activity; ion transport; membrane potentials; muscle cells; muscle pharmacology; neuropharmacology; ouabain; potassium ion; sodium ion; sodium potassium exchanging ATPase; squid; strophanthidin; voltage /patch clamp

The long-term objective of this project is to obtain an understanding of the mechanism of ion translocation by the electrogenic Na+/K+ pump at the molecular and physical-chemical level. The Na+/K+ pump is present in the surface membrane of nearly all animal cells, where it provides the predominant link between the cell's metabolic production of ATP and the maintenance of the Na+ ion and K+ ion electrochemical gradients. These gradients, in turn, underlie membrane potentials and electrical activity of both non-excitable and excitable cells and numerous Na+-coupled co- and counter-transport processes. The objective will be achieved by making measurements of steady-state and transient currents of radioisotopic unidirectional fluxes mediated by the Na+/K+ pump in its various models, and measurements of acid-induced, ATP- dependent, Na+/K+ pump-mediated (ouabain-sensitive) single-channel currents with six different voltage clamp techniques: 1) two- microelectrode voltage clamp of intact Xenopus oocytes, 2) cut-open oocytes, 3) patch clamp of oocytes, 4)reconstitution of oocyte vesicles in lipid bilayers, 5) simultaneous current and flux measurements in internally-dialyzed giant axons from squid, and 6) ultra-fast voltage clamp of pre-steady-state transient currents in internally dialyzed squid giant axons. The experimental plan is to exploit the strengths of each of these techniques: the intact oocyte for its stability, wide accessible voltage range, ease of external solution change, and convenience as a heterologous expression system; the cut-open oocyte for its high-speed voltage control and ease of cytoplasmic solution change; the patch clamp and bilayer techniques for their ability to resolve single channel current, and the squid axon for the ability to control both internal and external solutions, and to simultaneously measure radiotracer flux and voltage-clamp current, as well as the ability to produce ultra-fast voltage steps. The two specific aims of the project are: 1) to investigate the charge translocating steps in the reaction cycle of the Na+/K+ pump by measurements of pre-steady transient currents in Xenopus oocytes and squid giant axons to test the hypothesis that the transient currents reflect the sequential deocclusion and release of 3 Na+ ions to the extracellular side of the enzyme, and 2) to investigate the properties of acid-induced, ouabain-sensitive steady- state and single-channel current in oocyte membranes in K+-free solutions to test the hypothesis that a titrable amino acid is necessary for the maintenance of the Na+-occluded state of the Na+/K+ pump.

该项目的长期目标是获得了解 生电Na+/K+泵离子易位机制的研究 在分子和物理化学水平上。 Na+/K+泵是 存在于几乎所有动物细胞的表面膜中， 提供细胞代谢产物之间的主要联系 ATP 的生成以及 Na+ 离子和 K+ 离子电化学的维持 梯度。这些梯度反过来又是膜电位的基础 非兴奋性和兴奋性细胞的电活动 许多Na+耦合的共向和逆向运输过程。 目标 将通过测量稳态和瞬态来实现 由 Na+/K+ 介导的放射性同位素单向通量电流 各种型号的泵，以及酸诱导的 ATP- 的测量 依赖性、Na+/K+ 泵介导（哇巴因敏感）单通道 电流与六种不同的电压钳技术：1）二 完整爪蟾卵母细胞的微电极电压钳，2) 切开 卵母细胞，3) 卵母细胞膜片钳，4) 卵母细胞囊泡重建 在脂质双层中，5) 同时测量电流和通量 来自鱿鱼的内部透析巨型轴突，以及 6) 超快电压 内部透析中预稳态瞬态电流的钳位 鱿鱼巨大的轴突。 实验计划是发挥优势 这些技术中的每一种：完整卵母细胞的稳定性、广泛性 可访问的电压范围，易于更改外部解决方案，以及 作为异源表达系统的便利性；切开的卵母细胞 其高速电压控制和易于细胞质溶液变化； 膜片钳和双层技术的解析能力 单通道电流，以及鱿鱼轴突的控制能力 内部和外部解决方案，并同时测量 放射性示踪剂通量和电压钳电流，以及能力 产生超快的电压阶跃。 该项目的两个具体目标 是：1）研究反应中的电荷转移步骤 通过测量预稳态瞬态来确定 Na+/K+ 泵的循环 非洲爪蟾卵母细胞和鱿鱼巨轴突中的电流来检验这一假设 瞬态电流反映了顺序解除遮挡和 向酶的胞外侧释放 3 个 Na+ 离子，以及 2) 研究酸诱导的、哇巴因敏感的稳态的特性 无 K+ 状态下卵母细胞膜的单通道电流 检验可滴定氨基酸是否必要的假设的解决方案 用于维持 Na+/K+ 泵的 Na+ 吸留状态。