VOLTAGE-CLAMP STUDIES OF SODIUM PUMP CURRENT AND FLUX

该项目的目标是更好地了解 Na/K 泵反应循环中的电压依赖步骤。 四 具体目标是：1) 测量稳态 Na/K 泵电流-电压 泵运行的各种生电模式的关系 (I-V)，2) 测试Na/K泵的化学计量是否固定在3Na/2K并且是 在宽范围条件下与电压无关，3) 研究瞬态 Na/K 泵电流（泵电荷运动），以及 4) 测试泵是否 介导的Na/Na交换虽然是电中性的，但却是电压依赖性的。 至 实现这些目标，同时测量泵介导的变化 电流和通量，通过暴露于可逆泵抑制剂而产生 二氢洋地黄毒甙元 (H2DTG)，将以电压钳位、内部制造 透析鱿鱼巨型轴突。 对照实验已经证明 证明： 1) H2DTG 对离子电导没有直接影响； 2）当前和 22Na流出来自同一膜区域； 3）被动K的变化 由于停止泵时细胞外 [K] 的变化，电流为 很大程度上被 K 通道阻断剂所阻止； 4) 泵介导的Na/Na 通过添加高 [ATP] 和磷酸原最大限度地减少交换 磷酸烯醇丙酮酸和磷酸精氨酸进入透析液。 稳定- 状态 Na/K 泵 I-V，用于泵运行的正向和反向模式 将在不同条件下获得，因为之间的差异 在暴露于 H2DTG 之前和期间测量膜 I-V。 的 Na/K 泵的化学计量可以根据适当的情况进行研究 控制实验表明，H2DTG敏感的磁通和电流可以 等于 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。 瞬态泵浦电流的初步测量已在 在没有外部 K 的情况下，电压钳制爪蟾卵母细胞， 可能限制泵进行 Na/Na 交换。 来自这样的预测 测量，泵介导的电中性 Na/Na 交换是电压- 依赖性，可以通过评估电压依赖性在鱿鱼轴突中进行测试 H2DTG 敏感的 Na 从高 [ADP] 轴突流出到无 K 海水中。 进一步了解动物细胞的 Na/K 泵非常重要，因为 它维持了钠电化学梯度，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.