VOLTAGE-CLAMP STUDIES OF SODIUM PUMP CURRENT AND FLUX

本项目的目的是研究化学计量和 电压依赖性的钠泵的鱿鱼巨轴突使用 同时测量同位素22 Na流出或42 K流入， 生电泵电流 产电泵电流通过以下方式测量： 在添加可逆的 泵毒素二氢洋地黄毒苷（H2 DTG）或不可逆（鱿鱼） 哇巴因毒素 测量了保持电流的毒性敏感变化 使用稳定、低噪声的电压箝位装置。 的组成 细胞内轴浆由内分泌技术控制， 透析 该方法还允许使用放射性示踪剂用于 测量流入和流出。 通过使正常的离子 通过改变Na+和K+的梯度，可以使泵反向运行。 将进行实验以确定核苷酸变化的影响。 和离子组成对泵浦电流和 泵循环的化学计量。 已经做了一些实验， 证明反向运行的钠泵产生一个电 电流反向。 反向泵的大小 电流通过对应于以下区域的超极化而降低： 负斜率电导的电流-电压关系的 反向泵 实验还表明， 电流和通量被超极化抑制。 这些结果表明 膜电压可能是泵调节的重要因素 活动 泵浦钳位技术的有效性已得到验证， 证明了H_2DTG和哇巴因对被动膜无影响 电导和Na+通道或K+通道介导的测量 通量和电流在实验条件下是一致的， 只有单向离子流。 未来的实验将集中在 确定正向和反向电流的电流-电压关系， 泵操作的反向模式以及确定 泵的化学计量是固定的或可变的。 这些研究 对钠泵操作的机械影响， 在调节细胞体积和建立和 维持钠偶联转运的电化学梯度 在各种各样的动物和植物细胞中的过程。

The objective of this project is to investigate the stoichiometry and voltage dependence of the sodium pump of squid giant axons using simultaneous measurements of isotopic 22Na efflux or 42K influx and electrogenic pump current. The electrogenic pump current is measured by the change in holding current that occurs upon addition of the reversible pump toxin dihydrodigitoxigenin (H2DTG) or of the irreversible (in squid) toxin ouabain. The toxin-sensitive change in holding current is measured using a stable, low-noise voltage-clamp apparatus. The composition of the intracellular axoplasm is controlled by the technique of internal dialysis. This method also permits the use of radioactive tracers for the measurement of both influx and efflux. By steepening the normal ionic gradients for Na+ and K+, the pump can be made to run backwards. Experiments will be done to determine the effect of changes in nucleotide and ionic composition on the magnitude of the pump current and the stoichiometry of the pump cycle. Experiments have been done that demonstrate that the backward-running sodium pump produces an electrical current in the reverse direction. The magnitude of the reverse pump current is decreased by hyperpolarization corresponding to a region of negative slope conductance in the current-voltage relationship of the reverse pump. Experiments have also shown that both the forward pump current and flux are inhibited by hyperpolarization. These results suggest that membrane voltage may be an important factor in the regulation of pump activity. The validity of the pump-clamp technique has been verified by the demonstration that H2DTG and ouabain have no effect on passive membrane conductance and that measurements of Na+-channel or K+-channel mediated flux and current are in agreement under experimental conditions that allow only a unidirectional ion flow. Future experiments will focus on the determination of the current-voltage relationship of both the forward and reverse modes of pump operation and the determination of whether the stoichiometry of the pump is fixed or variable. These studies have mechanistic implications for the operation of the sodium pump which is important in the regulation of cell volume and in establishing and maintaining the electrochemical gradient for sodium-coupled transport processes in a wide variety of animal and plant cells.