VOLTAGE CLAMP STUDIES OF SODIUM PUMP CURRENT AND FLUX

这个项目的长期目标是获得一种理解 生电性Na/K泵离子转运机制的研究 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上，在那里它 在细胞的代谢产物之间提供了主要的联系 ATP与Na~+、K~+的电化学维持 渐变。这些梯度反过来又构成了膜电位和 不可兴奋和可兴奋细胞的电活动和 许多钠偶联的共转运和反转运过程。目标是 将通过对稳态和暂态进行测量来实现 Na/K介导的放射性同位素单向通量流 泵在其各种型号，并测量酸诱导，ATP- 依赖的Na/K泵介导(哇巴因敏感)单通道 具有六种不同电压钳位技术的电流：1)两- 非洲爪哇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)卵母细胞囊泡重构 在脂质双层中，5)同时测量电流和磁通 来自鱿鱼的内透析巨型轴突，以及超快电压 内透析中稳态前暂态电流的钳制 鱿鱼巨型轴突。实验计划是利用这些优势 这些技术中的每一种：完整的卵母细胞的稳定性，宽 可访问的电压范围，易于更换外部解决方案，以及 方便作为异源表达系统；切开的卵母细胞用于 其高速的电压控制和胞浆溶液的易变性； 膜片钳和双层技术对其分辨能力的影响 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制电流，以及 产生超快的电压阶跃。该项目的两个具体目标 主要包括：1)研究反应中的电荷转移步骤 用定常前暂态测量实现钠钾泵的循环 非洲爪哇卵母细胞和鱿鱼巨轴突中的电流来验证这一假说 暂态电流反映的是顺序脱合和 3个Na离子释放到酶的胞外侧，以及2) 为了研究酸诱导、哇巴因敏感的稳定- 无钾条件下卵母细胞膜的状态电流和单通道电流 检验可滴定氨基酸是必需的假设的解决方案 以维持钠钾泵的钠封闭状态。

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.