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: 2264669
• 负责人: ROBERT F RAKOWSKI
• 金额: $ 21.15万
• 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 1998-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2264669
• 关键词: Xenopus; Xenopus oocyte; animal genetic material tag; axon; complementary DNA; complementary RNA; enzyme activity; enzyme mechanism; ion transport; membrane potentials; neural conduction; potassium; radionuclides; rubidium; sodium; sodium potassium exchanging ATPase; squid; voltage /patch clamp

The long-term objective of this project is to obtain a greater understanding of the transport mechanism of the Na/K pump by identifying and characterizing the charge translocating, and hence voltage dependent, steps in the Na/K pump reaction cycle. The Na/K pump is present in the surface membrane of nearly all animal cells, where it provides the link between the cell's production of ATP and the maintenance of the Na 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 Na/K pump is also the receptor for a clinically important class of drugs, cardiotonic steroids, used in the treatment of heart failure and certain cardiac arrhythmias. The objective will be achieved by making measurements of steady-state and transient currents, and of radioisotopically-labeled unidirectional fluxes mediated by the Na/K pump in its various modes, in three voltage clamped preparations: intact Xenopus oocytes, cut-open oocytes, and internally-dialyzed, giant axons from squid. The experimental plan is to exploit the strengths of each of these preparations: 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 squid axon for the ability to control both internal and external solutions, and to simultaneously measure radiotracer flux and voltage-clamp current. The four specific aims of the project are: l) to examine the external [Na] and [K] dependence of the steady-state pump current-voltage (I-V) relationship in intact oocytes and in squid axons, 2) to examine the internal [Na] and [K] dependence of the steady-state pump I-V relationship in cut-open oocytes and in squid axons, 3) to examine the internal and external ion dependence of 22Na and 42K or 86Rb efflux mediated by the Na/K pump in its various transport modes in internally-dialyzed, voltage clamped squid giant axons, and 4) to measure and analyze voltage-jump-induced, pre-steady-state, transient currents under Na/Na exchange conditions using the cut-open oocyte voltage-clamp technique.

该项目的长期目标是获得更大的 通过识别了解钠/钾泵的传输机制 并且表征电荷移位，并且因此是电压依赖的， Na/K泵反应循环中的步骤。Na/K泵存在于 几乎所有动物细胞的表面膜，在那里它提供了连接 细胞产生ATP和维持Na和K 离子电化学梯度这些梯度，反过来， 非兴奋性和兴奋性的电位和电活动 细胞和许多钠耦合的共同和反运输过程。的 Na/K泵也是临床上一类重要药物的受体， 强心类固醇，用于治疗心力衰竭和某些 心律失常这一目标将通过测量来实现 稳态和瞬态电流，以及放射性同位素标记的 单向通量介导的钠/钾泵在其各种模式， 三种电压钳夹制备物：完整的非洲爪蟾卵母细胞，切开 卵母细胞和内部透析的鱿鱼巨大轴突。实验 计划是利用这些准备工作中每一个的优势： 卵母细胞的稳定性，宽的可达电压范围，易于外部 解决方案的变化，并方便作为异源表达系统; 切割开放的卵母细胞，其高速电压控制和细胞质的容易 解决方案的变化;鱿鱼轴突的能力，以控制内部 和外部溶液，并同时测量放射性示踪剂通量和 电压箝位电流该项目的四个具体目标是： 检查稳态泵的外部[Na]和[K]依赖性 完整卵母细胞和鱿鱼轴突中的电流-电压（I-V）关系， 2)研究稳态的内部[Na]和[K]依赖性， 在切开的卵母细胞和鱿鱼轴突中的泵I-V关系，3） 检查22 Na和42 K或86 Rb的内部和外部离子依赖性 Na/K泵在其各种转运模式中介导的外排， 内部透析，电压钳乌贼巨大的轴突，和4）测量 并分析电压跳变引起的预稳态瞬态电流 在Na/Na交换条件下，使用切开的卵母细胞电压钳 法