VOLTAGE-CLAMP STUDIES OF SODIUM PUMP CURRENT AND FLUX

这个项目的目标是研究化学计量学和 乌贼巨型轴突钠泵的电压依赖性 同位素~(22)Na流出或~(42)K流出的同时测量 生电泵电流。产生电泵的电流通过以下方式测量 当增加可逆电流时，保持电流的变化。 泵毒素二氢洋地黄毒素(H_2DTG)或不可逆性毒素(在鱿鱼中) 哇巴因毒素。测量了保持电流对毒素敏感的变化 使用稳定、低噪声的电压钳位装置。这份报告的组成 细胞内轴浆由内分泌技术控制 透析。这种方法还允许将放射性示踪剂用于 流入和流出的测量。通过使正常离子变陡 对于钠和钾的梯度，可以使泵向后运行。 将进行实验以确定核苷酸变化的影响 以及离子组成与泵电流的大小和 泵循环的化学计量。已经做了这样的实验 证明向后运行的钠泵产生一种电能 电流方向相反。反向泵的大小 电流通过对应于以下区域的超极化来减小 电流-电压关系中的负斜率电导 反转泵。实验还表明，前向泵 电流和磁通受到超极化的抑制。这些结果表明 膜电压可能是泵调节的一个重要因素。 活动。泵-夹具技术的有效性得到了验证 H_2DTG和哇巴因对钝化膜无影响的研究 钠通道或钾通道介导性电导及测量 磁通和电流在允许的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 正向和反向电流-电压关系的确定 泵运行的反向模式和确定是否 泵的化学计量比是固定的或可变的。这些研究已经 对钠泵运行的机械影响 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进行加工。

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.