Division of Nephrology, Department of Medicine and Graduate School of Biomedical Sciences, The Icahn School of Medicine at Mount Sinai, New York, New York, United States of America.
PLoS One. 2013 May 23;8(5):e60836. doi: 10.1371/journal.pone.0060836. Print 2013.
The planar lipid bilayer technique has a distinguished history in electrophysiology but is arguably the most technically difficult and time-consuming method in the field. Behind this is a lack of experimental consistency between laboratories, the challenges associated with painting unilamellar bilayers, and the reconstitution of ion channels into them. While there has be a trend towards automation of this technique, there remain many instances where manual bilayer formation and subsequent membrane protein insertion is both required and advantageous. We have developed a comprehensive method, which we have termed "wicking", that greatly simplifies many experimental aspects of the lipid bilayer system. Wicking allows one to manually insert ion channels into planar lipid bilayers in a matter of seconds, without the use of a magnetic stir bar or the addition of other chemicals to monitor or promote the fusion of proteoliposomes. We used the wicking method in conjunction with a standard membrane capacitance test and a simple method of proteoliposome preparation that generates a heterogeneous mixture of vesicle sizes. To determine the robustness of this technique, we selected two ion channels that have been well characterized in the literature: CLIC1 and α-hemolysin. When reconstituted using the wicking technique, CLIC1 showed biophysical characteristics congruent with published reports from other groups; and α-hemolysin demonstrated Type A and B events when threading single stranded DNA through the pore. We conclude that the wicking method gives the investigator a high degree of control over many aspects of the lipid bilayer system, while greatly reducing the time required for channel reconstitution.
平面脂质双层技术在电生理学中有着悠久的历史，但它是该领域技术上最困难和最耗时的方法。这背后是实验室之间缺乏实验一致性，单分子层脂质体绘画的挑战，以及离子通道的重新构成。虽然这种技术已经有了自动化的趋势，但在许多情况下，手动双层形成和随后的膜蛋白插入仍然是必需的和有利的。我们开发了一种综合方法，我们称之为“吸吮”，它极大地简化了脂质双层系统的许多实验方面。吸吮法允许在几秒钟内将离子通道手动插入平面脂质双层，而无需使用搅拌棒或添加其他化学物质来监测或促进脂蛋白体的融合。我们将吸吮法与标准膜电容测试以及一种简单的脂蛋白体制备方法结合使用，该方法生成了大小不均的囊泡混合物。为了确定这种技术的稳健性，我们选择了两种在文献中得到很好描述的离子通道：CLIC1 和 α-溶血素。当使用吸吮技术重新构成时，CLIC1 表现出与其他组发表的报告一致的生物物理特征；并且当单链 DNA 通过孔时，α-溶血素表现出 A 型和 B 型事件。我们得出结论，吸吮法使研究人员能够高度控制脂质双层系统的许多方面，同时大大减少了通道重建所需的时间。
