VOLTAGE CLAMPING OF XENOPUS-LAEVIS OOCYTES UTILIZING AGAROSE-CUSHION ELECTRODES
VOLTAGE CLAMPING OF XENOPUS-LAEVIS OOCYTES UTILIZING AGAROSE-CUSHION ELECTRODES
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DOI：
10.1007/bf00388310
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发表时间：
1994-03-01
影响因子：
4.5
通讯作者：
DASCAL, N
中科院分区：
文献类型：
--
作者：
SCHREIBMAYER, W;LESTER, HA;DASCAL, N
在完整的卵母细胞的南非爪蛙非洲爪蟾的表达的离子通道的双电极电压钳已被完善，以允许稳定，低电阻的电访问的胞质溶胶（50-800 k Ω）。玻璃微电极在其尖端填充有1%琼脂糖垫，以防止KCl泄漏（琼脂糖垫电极）。将这些电极插入X。在1小时以上的时间内，非洲乳鼠卵母细胞产生稳定的制备物，具有1-4 M Ω的稳定输入抗性。此外，描述了电压钳电路（充电补偿器）的简单修改，其增加了用于膜电位的差分记录的布置的灵活性，以便减去串联电阻两端的电压降。其结果是，用传统的双电极布置的电压钳的实际可达到的速度有相当大的提高。在模拟卵母细胞和电极的等效电路上测试充电补偿器的性能。此外，琼脂糖垫电极和充电补偿器的组合进行了测试表达摇床H4电流的卵母细胞。电压钳电路的保真度也通过测量膜电位与连接到差分放大器的额外的独立的微电极，独立的双电极电压钳系统。该系统可用于X射线离子通道的研究。需要长期记录和/或测量大的、快速的离子电流的卵母细胞。
Two-electrode voltage clamping of expressed ion channels in intact oocytes of the South African clawed frog Xenopus laevis has been refined to allow stable, low-resistance electrical access to the cytosol (50-800 k Omega). Glass microelectrodes were filled with a cushion of 1% agarose at their tips to prevent KCl leakage (agarose-cushion electrodes). Insertion of these electrodes into X. laevis oocytes yielded stable preparations for periods of more than Ih with a stable input resistance of 1-4 M Omega. Furthermore, a simple modification of the voltage-clamp circuit (charging compensator) is described that increases the flexibility of arrangements for differential recording of the membrane potential in order to subtract voltage drops across a series resistance. The result is a considerable increase in the practically attainable speed of the voltage clamp with the conventional two-electrode arrangement. The performance of the charging compensator was tested on an equivalent circuit that simulates the oocyte and electrodes. In addition, the combination of agarose-cushion electrodes and the charging compensator was tested on oocytes expressing Shaker H4 currents. The fidelity of the voltage-clamp circuit was also verified by measuring the membrane potential with additional independent microelectrodes connected to a differential amplifier, independent of the two-electrode voltage clamp system. The system described here will be useful for ion channel studies in X. laevis oocytes requiring long-term recordings and/or measurements of large, fast ion currents.