Qin D Y, Noma A
Department of Physiology, Faculty of Medicine, Kyushu University, Fukuoka, Japan.
Am J Physiol. 1988 Oct;255(4 Pt 2):H980-4. doi: 10.1152/ajpheart.1988.255.4.H980.
A new method was developed to instantaneously replace the solution on the inner side of an inside-out membrane patch in order to measure time courses with which active substances acted on single ionic channels. Inside-out membrane patches were isolated from single ventricular cells of the guinea pig heart. The recording bath consisted of two chambers separated by a partition having a narrow slit. Mixing of two test solutions through this slit was prevented by filling it with paraffin oil. The pipette tip with a tightly sealed inside-out membrane patch was moved through the oil from one solution to the other so that the pipette tip was instantaneously exposed to a new solution. When the pipette tip was jumped between different K+ concentrations, the leak current through the membrane patch increased or decreased with a half time of 6.3 +/- 3.0 ms (n = 15). The amplitude of single K+ channel currents changed to a new steady level within approximately 20 ms. These time courses were well explained by diffusion of K+ in the dead space between the pipette tip opening and the membrane patch. An application of this method to the ATP-regulated K+ channel revealed a latent period of 1-2 s before the channel started its activity after the instantaneous removal of ATP, whereas no obvious latency was observed in the rapid suppression of the channel, which was completed in 100-300 ms after reapplying ATP.
为了测量活性物质作用于单个离子通道的时间进程，人们开发了一种新方法，可即时替换外翻式膜片内侧的溶液。外翻式膜片取自豚鼠心脏的单个心室细胞。记录浴槽由两个腔室组成，中间由一个带有狭窄缝隙的隔板隔开。通过向缝隙中填充石蜡油来防止两种测试溶液通过该缝隙混合。将带有紧密密封的外翻式膜片的移液管尖端从一种溶液穿过油层移至另一种溶液，使得移液管尖端瞬间暴露于新溶液中。当移液管尖端在不同的K⁺浓度之间切换时，通过膜片的漏电流以6.3±3.0毫秒的半衰期增加或减少（n = 15）。单个K⁺通道电流的幅度在约20毫秒内变为新的稳定水平。这些时间进程可以通过K⁺在移液管尖端开口与膜片之间的死腔内的扩散得到很好的解释。将该方法应用于ATP调节的K⁺通道时发现，在瞬间去除ATP后，通道开始活动前有1 - 2秒的潜伏期，而在重新施加ATP后100 - 300毫秒内通道迅速受到抑制，未观察到明显的潜伏期。
