Stühmer W, Conti F, Stocker M, Pongs O, Heinemann S H
Max-Planck-Institut für biophysikalische Chemie, Göttingen, Federal Republic of Germany.
Pflugers Arch. 1991 May;418(4):423-9. doi: 10.1007/BF00550881.
The Xenopus oocyte expression system in combination with patch-clamp techniques allows the measurement of ionic currents from a single class of genetically engineered ion channels. Ionic currents in the nanoampere range from oocytes injected with cRNA, corresponding to potassium channels, can be recorded in the inside-out patch configuration. These recordings have a high time resolution at low background noise. Substitution of impermeant ions for potassium and blocking of the channel conductance with tetraethylammonium allows the recording of potassium gating currents, Ig, which is hampered in natural excitable cells by the simultaneous presence of sodium channels and a variety of different potassium channels. The "on" transients, Ig(on), are fast and can have amplitudes of up to several tens of pA. Upon repolarization to -100 mV after small depolarizations, "off" gating currents, Ig(off)g, which reverse most of the "on" charge displacement, Q(on), within 1 ms, are readily observed. However, this fast recovery of the gating charge is drastically reduced upon increasing the amplitude of the depolarizing pulse. In contrast to sodium channels, this temporary charge immobilization is complete within a few milliseconds at positive membrane potentials. Furthermore, there seems to be no direct correlation between charge immobilization and inactivation because the same phenomenon occurs for channels that do not inactivate.
非洲爪蟾卵母细胞表达系统与膜片钳技术相结合,能够测量来自单一类别的基因工程离子通道的离子电流。在膜片外翻配置中,可以记录注射了对应钾通道的cRNA的卵母细胞中纳安级别的离子电流。这些记录在低背景噪声下具有较高的时间分辨率。用不可渗透离子替代钾离子并用四乙铵阻断通道电导,可以记录钾离子门控电流Ig,而在天然可兴奋细胞中,由于同时存在钠通道和多种不同的钾通道,这一过程受到阻碍。“开启”瞬变电流Ig(on)很快,幅度可达数十皮安。在小幅度去极化后复极化至 -100 mV时,很容易观察到“关闭”门控电流Ig(off),它在1毫秒内使大部分“开启”电荷位移Q(on)反转。然而,当增加去极化脉冲的幅度时,门控电荷的这种快速恢复会急剧降低。与钠通道不同,这种暂时的电荷固定在正膜电位下几毫秒内就会完成。此外,电荷固定与失活之间似乎没有直接关联,因为对于不发生失活的通道也会出现相同的现象。