VOLTAGE CLAMP STUDIES OF SODIUM PUMP CURRENT AND FLUX

本项目的长期目标是了解 离子迁移的机制，通过产电Na+/K+泵 在分子和物理化学水平上。 Na+/K+泵 存在于几乎所有动物细胞的表面膜中， 提供了细胞代谢产物之间的主要联系 ATP和维持Na+和K+离子的电化学 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+耦合的协同和逆向转运过程。 客观 将通过测量稳态和瞬态 Na+/K+介导的放射性同位素单向通量电流 泵在其各种模型，和测量酸诱导，ATP- 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位技术：1）两个- 完整非洲爪蟾卵母细胞的微电极电压钳，2）切开 卵母细胞，3）卵母细胞的膜片钳，4）卵母细胞囊泡的重建 在脂质双层中，5）同时测量 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时电流的钳位 乌贼巨大的轴突 实验计划是利用 这些技术中的每一种：完整的卵母细胞的稳定性， 可访问的电压范围，易于更换外部解决方案，以及 作为异源表达系统的便利性;用于 电压控制速度快，细胞质溶液易更换; 膜片钳和双层技术的能力，以解决 单通道电流和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 产生超快的电压阶跃。 该项目的两个具体目标 研究了反应中的电荷转移步骤 通过测量稳态前瞬态的Na ~+/K ~+泵循环 电流在非洲爪蟾卵母细胞和鱿鱼巨大的轴突，以检验这一假设 瞬时电流反映了连续的去闭塞， 释放3个Na+离子到酶的胞外侧，和2） 研究酸诱导的、哇巴因敏感的稳定的 无钾条件下卵母细胞膜稳态和单通道电流 测试可滴定氨基酸是必需的假设的解决方案 用于维持Na+/K+泵的Na+吸留状态。

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.