VOLTAGE-CLAMP STUDIES OF SODIUM PUMP CURRENT AND FLUX

这个项目的目的是为了更好地了解 Na/K泵反应周期中的电压依赖性步骤(S)。四 具体目标是：1)测量稳态Na/K泵电流-电压 泵运行的各种发电模式的关系(I-V)，2) 测试Na/K泵的化学计量比是否固定在3nA/2K 在宽范围条件下与电压无关，3)调查暂态 Na/K泵浦电流(泵浦电荷运动)，以及4)测试是否泵浦- 介导的Na/Na交换，虽然是电中性的，但却是电压依赖的。至 实现这些目标，同时测量泵调节的变化 电流和磁通，由暴露在可逆泵抑制剂中产生 二氢洋地黄毒素(H_2DTG)，将在电压钳制，内部- 透析的鱿鱼巨型轴突。对照实验已经证明 即：1)H_2DTG对离子电导没有直接影响；2)电流和 22Na外流来自相同的膜面积；3)被动钾的变化 由于停止泵时细胞外[K]的变化，电流是 主要由钾通道阻滞剂阻止；4)泵介导的Na/Na 通过添加高[ATP]和磷酸根来最小化交换 磷酸烯醇式丙酮酸和磷酸精氨酸进入透析液。稳定- 说明Na/K泵正向和反向运行模式的I-V 将在不同条件下获得的差值 在暴露于H_2DTG之前和过程中测量膜I-V。这个 可以研究Na/K泵的化学计量比，因为适当的 控制实验表明，对H_2DTG敏感的磁通和电流可以 等同于Na/K泵的流量和电流，从而用于计算 在泵不输送其他离子的情况下，Na/K转运的比率。 对瞬变泵浦电流进行了初步测量。 电压钳制非洲爪哇卵母细胞在没有外钾的情况下， 据推测，泵仅限于钠/钠交换。来自这样的预测 测量表明，泵浦介导的电中性钠/钠交换是电压-钠交换。 依赖，可以通过评估电压依赖在鱿鱼轴突中进行测试 H_2DTG敏感的钠从高[ADP]轴突流出到无钾海水中。 进一步了解动物细胞的Na/K泵很重要，因为 它保持钠的电化学梯度，这是必不可少的 兴奋性和钠偶联转运过程。

The objective of this project is to obtain a greater understanding of the voltage dependent step(s) in the reaction cycle of the Na/K pump. Four specific aims are: 1) measure steady-state Na/K pump current-voltage relationships (I-Vs) for various electrogenic modes of pump operation, 2) test whether the stoichiometry of the Na/K pump is fixed at 3Na/2K and is voltage-independent over wide-ranging conditions, 3) investigate transient Na/K pump currents (pump charge movement), and 4) test whether pump- mediated Na/Na exchange, though electroneutral, is voltage-dependent. To achieve these aims, simultaneous measurements of changes in pump-mediated current and flux, produced by exposure to the reversible pump inhibitor dihydrodigitoxigenin (H2DTG), will be made in voltage-clamped, internally- dialyzed squid giant axons. Control experiments have already demonstrated that: 1) H2DTG has no direct effect on ionic conductances; 2) current and 22Na efflux derive from the same membrane area; 3) changes in passive K current, due to changes of extracellular [K] on stopping the pump, are largely prevented by K channel-blocking agents; 4) pump-mediated Na/Na exchange has been minimized by adding high [ATP] and the phosphagens phosphoenolpyruvate and phosphoarginine, to the dialysis fluid. Steady- state Na/K pump I-Vs for the forward and reverse modes of pump operation will be obtained under various conditions as the difference between membrane I-Vs measured before and during exposure to H2DTG. The stoichiometry of the Na/K pump can be investigated since appropriate control experiments have shown that H2DTG-sensitive flux and current can be equated with Na/K pump flux and current, and thus used to calculate the ratio of Na to K transport, provided the pump transports no other ion. Preliminary measurements of transient pump currents have been made in voltage-clamped Xenopus oocytes in the absence of external K, which presumably limits the pump to Na/Na exchange. A prediction from such measurements, that pump-mediated electroneutral Na/Na exchange is voltage- dependent, can be tested in squid axons by assessing the voltage dependence of H2DTG-sensitive Na efflux from high-[ADP] axons into K-free seawater. Further understanding of the Na/K pump of animal cells is important since it maintains the Na electrochemical gradient which is essential for excitability and for Na-coupled transport processes.