Shuba Y M, Naidenov V G, Morad M
Department of Pharmacology, Georgetown University Medical Center, 3900 Reservoir Road, NW, Washington DC 20007, USA.
Pflugers Arch. 1996 Jul;432(3):562-70. doi: 10.1007/s004240050170.
In this report we present a description of a modified version of the "glass-funnel" technique for the recording of membrane currents and intracellular perfusion of Xenopus laevis oocytes. The technique is based on the ability of the devitellinated oocyte to form a high-resistance seal with the glass, permitting separation of the oocyte into two, i.e., extra- and intracellular, compartments. The technique is fairly simple to use, provides a much higher clamp speed compared to the double-microelectrode voltage-clamp technique, and allows effective control of the composition of the intracellular milieu. To elucidate the performance of the technique with respect to various membrane currents we present data relating to the recording of Ca-channel currents expressed in X. laevis oocytes by means of mRNA extracted from the rat cerebellum and heart, as well as currents induced by cRNA for the skeletal muscle micro1 Na+ channel and the dog heart NCX1 Na+-Ca2+ exchanger. Due to effective elimination of intra- and extracellular Cl- it became possible to measure not only Ba2+ but also Ca2+ current through the expressed Ca channels, and to record the activity of the Na+-Ca2+ exchanger following dialysis of the oocyte with high-Ca2+ intracellular solutions. Corresponding currents showed properties identical to those obtained with other techniques, suggesting the adequacy of the glass-funnel technique for critical analysis of membrane ionic currents in Xenopus oocytes.
在本报告中，我们描述了一种用于记录非洲爪蟾卵母细胞膜电流和进行细胞内灌流的“玻璃漏斗”技术的改良版本。该技术基于脱卵黄卵母细胞与玻璃形成高电阻封接的能力，从而可将卵母细胞分隔为两个部分，即细胞外和细胞内部分。该技术使用起来相当简单，与双微电极电压钳技术相比，钳制速度要高得多，并且能够有效控制细胞内环境的成分。为了阐明该技术在记录各种膜电流方面的性能，我们展示了有关通过从大鼠小脑和心脏提取的mRNA在非洲爪蟾卵母细胞中表达的钙通道电流记录的数据，以及由骨骼肌微1钠通道和犬心脏NCX1钠钙交换体的cRNA诱导的电流记录数据。由于有效消除了细胞内和细胞外的氯离子，不仅可以测量通过表达的钙通道的钡离子电流，还可以测量钙离子电流，并且在用高钙细胞内溶液透析卵母细胞后记录钠钙交换体的活性。相应的电流表现出与其他技术获得的电流相同的特性，这表明玻璃漏斗技术足以对非洲爪蟾卵母细胞中的膜离子电流进行关键分析。
