VOLTAGE CLAMP STUDIES OF SODIUM PUMP CURRENT AND FLUX

这个项目的长期目标是获得更大的 通过识别认识钠钾泵的输运机理 并描述了电荷的转移，因此与电压有关， 在钠/钾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，在那里它提供了联系 细胞产生三磷酸腺苷与维持钠、钾之间的关系 离子电化学梯度。这些梯度依次位于膜的下面。 不可兴奋和可兴奋的电位和电活动 细胞和许多钠偶联的共转运和反向转运过程。这个 Na/K泵也是一类临床重要药物的受体， 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 稳态和暂态电流，以及放射性同位素标记的 Na/K泵在其不同模式下调节的单向通量 三种电压钳制方法：完整的非洲爪哇卵母细胞，切开 卵母细胞，以及内部透析的来自鱿鱼的巨大轴突。实验性的 计划是利用每一种准备的优势：完好无损的 卵母细胞因其稳定性好、可及电压范围广、易外接 溶液的变化，以及作为异源表达系统的方便性； 切割卵母细胞的高速电压控制和易细胞质 解决方案改变；对于鱿鱼轴突的内部控制能力 和外部解决方案，并同时测量放射性示踪剂通量和 电压钳位电流。该项目的四个具体目标是：L)到 考察稳态泵的外[Na]和[K]相关性 完整卵母细胞和鱿鱼轴突的电流-电压关系， 2)考察稳态的[Na]和[K]的内在依赖性 切开卵母细胞和鱿鱼轴突的泵I-V关系，3) 研究22Na和42K或86Rb的内部和外部离子依赖性 Na/K泵在不同转运方式下介导的外流 内透析、电压钳制的鱿鱼巨型轴突，以及4)测量 并分析了电压跳跃感应、稳态前、暂态电流 在Na/Na交换条件下使用切开卵母细胞电压钳 技术。

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.