title: Wicking: a rapid method for manually inserting ion channels into planar lipid bilayers.
abstract: 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.
authors.affiliation: 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.
authors.forename: Justin A
authors.initials: JA
authors.lastname: Costa
authors.forename: Dac A
authors.initials: DA
authors.lastname: Nguyen
authors.forename: Edgar
authors.initials: E
authors.lastname: Leal-Pinto
authors.forename: Ronald E
authors.initials: RE
authors.lastname: Gordon
authors.forename: Basil
authors.initials: B
authors.lastname: Hanss
mesh_terms: D000465:Algorithms; D001426:Bacterial Proteins / Q000737:chemistry*; D002197:Capillary Action; D018118:Chloride Channels / Q000037:antagonists & inhibitors / Q000737:chemistry*; D025802:Electric Capacitance; D006016:Glycolates / Q000737:chemistry; D057809:HEK293 Cells; D006460:Hemolysin Proteins / Q000737:chemistry*; D006801:Humans; D015640:Ion Channel Gating; D007473:Ion Channels / Q000737:chemistry; D008051:Lipid Bilayers / Q000737:chemistry*; D008081:Liposomes / Q000737:chemistry / Q000648:ultrastructure; D008564:Membrane Potentials; D010714:Phosphatidylethanolamines / Q000737:chemistry; D010718:Phosphatidylserines / Q000737:chemistry
chemical_list: D001426:Bacterial Proteins; C501966:CLIC1 protein, human; D018118:Chloride Channels; D006016:Glycolates; D006460:Hemolysin Proteins; D007473:Ion Channels; D008051:Lipid Bilayers; D008081:Liposomes; D010714:Phosphatidylethanolamines; D010718:Phosphatidylserines; C483858:phosphatidylethanolamine; C015765:MK 473