VOLTAGE-CLAMP STUDIES OF SODIUM PUMP CURRENT AND FLUX

该项目的目标是更好地了解 Na/K泵的反应循环中的电压依赖性步骤。 四 具体目的是：1）测量稳态Na/K泵电流-电压 泵操作的各种生电模式的关系（I-Vs），2） 测试Na/K泵的化学计量是否固定在3 Na/2K， 在宽范围条件下与电压无关，3）研究瞬态 Na/K泵电流（泵电荷移动），以及4）测试泵- 介导的Na/Na交换虽然是电中性的，但却是电压依赖性的。 到 实现这些目标，同时测量泵介导的 暴露于可逆泵抑制剂产生的电流和通量 二氢洋地黄毒苷（H2 DTG），将在电压钳位，内部- 透析了鱿鱼的巨大轴突 对照实验已经证明 结果表明：1）H2 DTG对离子电导没有直接影响; 2）电流和 22 Na流出来自同一膜面积; 3）被动钾离子浓度的变化 电流，由于细胞外[K]的变化停止泵，是 钾通道阻断剂在很大程度上阻止; 4）泵介导的Na/Na 通过添加高[ATP]和磷酸盐， 磷酸烯醇丙酮酸和磷酸精氨酸。 稳住- 用于泵操作的正向和反向模式的状态Na/K泵I-Vs 将在各种条件下获得， 在暴露于H2 DTG之前和期间测量膜I-Vs。 的 可以研究Na/K泵的化学计量，因为适当的 控制实验表明，H2 DTG敏感通量和电流可以 等同于Na/K泵通量和电流，因此用于计算 Na与K传输的比率，前提是泵不传输其他离子。 瞬态泵电流的初步测量已在 电压钳爪蟾卵母细胞在没有外部K， 可能将泵限制于Na/Na交换。 一个这样的预测 测量，泵介导的电中性Na/Na交换是电压- 依赖，可以通过评估电压依赖性在鱿鱼轴突中进行测试 从高[ADP]轴突到无钾海水的H2 DTG敏感的Na流出。 进一步了解动物细胞的Na/K泵是重要的，因为 它保持了钠的电化学梯度，这对于 兴奋性和钠耦合运输过程。

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.