Larsen E H, Price E M, Gabriel S E, Stutts M J, Boucher R C
Cystic Fibrosis/Pulmonary Research and Treatment Center, School of Medicine, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7248, USA.
Pflugers Arch. 1996 Jul;432(3):528-37. doi: 10.1007/s004240050166.
The Sf9 insect Spodoptora frugiperda cell line was used for heterologous expression of the cloned human cystic fibrosis transmembrane conductance regulator (CFTR) cDNA, or the cloned beta-galactosidase gene, using the baculovirus Autographa califonica as the infection vector. Using application of the patch-clamp technique, evidence for functional expression of CFTR was obtained according to the following three criteria. Firstly, whole-cell currents recorded 2 days after infection with CFTR revealed a statistically significant increase of membrane conductance, approximately 25 times above that of mock-infected control cells, with the reversal potential of the major current component being governed by the chloride equilibrium potential (ECl). Secondly, in contrast to uninfected cells and cells infected with beta-galactosidase, the membrane conductance to chloride of CFTR-injected cells was stimulated by cytosolic adenosine 3',5'-cyclic monophosphate (cAMP), which was raised by exposing the cells to 10 microM forskolin. Thirdly, recordings of currents through single channels in excised outside-out membrane patches of CFTR-infected cells revealed channels which were clearly different from the native insect chloride channel. Excised outside-out patches of CFTR-infected and forskolin-stimulated cells exhibited wave-like gating kinetics of well-resolved current transitions. All-point Gaussian distributions revealed contributions from several (five to nine) identical channels. Such channels, in excised outside-out patches, studied with a pipette [Cl-] = 40 mM and a bath [Cl-] = 150 mM, rectified the current in agreement with simple electrodiffusion and with a single-channel Goldman-Hodgkin-Katz permeability, PCl = 1. 34 x 10(-14) +/- 0.23 x 10(-14 )cm3/s (n = 5), corresponding to a physiological single-channel conductance of 2.8 +/- 0.5 pS (VM = ECl) and a limiting conductance, gamma150/150, = 7.7 +/- 1.3 pS ([Cl-]Bath = [Cl-]Cell = 150 mM). Currents recorded from multichannel excised outside-out patches could shift from the above mode of resolvable unitary conductance transitions to one which was too fast to reveal the dwell-times of closed and open states. During periods characterized by noisy currents, the variance (sigma2) of current fluctuations about their stationary mean value depicted a U-shaped function of membrane potential, with a minimum value at a pipette potential where the chloride current was shown to be zero. Thus, it can be concluded that the current fluctuations are caused by fast gating of channels specific for chloride ions. Switching back and forth between the two gating modes of clusters of chloride channels occurred from moment to moment in excised patches when the membrane potential was held at a constant value indicating cooperative gating as a result of interaction between neighbouring chloride channels.
使用杆状病毒苜蓿银纹夜蛾多角体病毒作为感染载体，在草地贪夜蛾 Sf9 昆虫细胞系中进行克隆的人类囊性纤维化跨膜传导调节因子（CFTR）cDNA 或克隆的β-半乳糖苷酶基因的异源表达。应用膜片钳技术，根据以下三个标准获得了 CFTR 功能表达的证据。首先，感染 CFTR 后 2 天记录的全细胞电流显示膜电导有统计学上的显著增加，比模拟感染的对照细胞高约 25 倍，主要电流成分的反转电位由氯离子平衡电位（ECl）决定。其次，与未感染细胞和感染β-半乳糖苷酶的细胞相比，CFTR 注射细胞对氯离子的膜电导受到胞质 3',5'-环磷酸腺苷（cAMP）的刺激，通过将细胞暴露于 10 μM 福斯高林可提高 cAMP 水平。第三，在 CFTR 感染细胞的外翻式膜片上单通道电流记录显示通道明显不同于天然昆虫氯离子通道。CFTR 感染并经福斯高林刺激的细胞的外翻式膜片表现出分辨良好的电流跃迁的波状门控动力学。全点高斯分布显示来自几个（五到九个）相同通道的贡献。在使用吸管[Cl-] = 40 mM 和浴液[Cl-] = 150 mM 研究的外翻式膜片中，这种通道使电流整流，符合简单的电扩散和单通道戈德曼-霍奇金-卡茨渗透率，PCl = 1.34×10(-14)±0.23×10(-14) cm3/s（n = 5），对应于生理单通道电导 2.8±0.5 pS（VM = ECl）和极限电导，γ150/150 = 7.7±1.3 pS（[Cl-]浴液 = [Cl-]细胞 = 150 mM）。从多通道外翻式膜片记录的电流可以从上述可分辨的单位电导跃迁模式转变为一种太快而无法揭示关闭和开放状态停留时间的模式。在以噪声电流为特征的时期，电流围绕其稳定平均值的波动方差（σ2）描绘了膜电位的 U 形函数，在吸管电位处有最小值，此时氯离子电流显示为零。因此，可以得出结论，电流波动是由氯离子特异性通道的快速门控引起的。当膜电位保持在恒定值时，在切除的膜片中，氯离子通道簇的两种门控模式之间会瞬间来回切换，这表明相邻氯离子通道之间的相互作用导致了协同门控。
