Wicking: A Rapid Method for Manually Inserting Ion Channels into Planar Lipid Bilayers

Authors

Justin A. Costa, The Icahn School of Medicine at Mount Sinai
Dac A. Nguyen, The Icahn School of Medicine at Mount Sinai
Egdar Leal-Pinto, Virginia Commonwealth University
Ronald E. Gordon, The Icahn School of Medicine at Mount Sinai
Basil Hanss, The Icahn School of Medicine at Mount Sinai

Document Type

Article

Original Publication Date

2013

Journal/Book/Conference Title

PLOS ONE

Volume

8

DOI of Original Publication

10.1371/journal.pone.0060836

Comments

Originally Published at http://dx.doi.org/10.1371/journal.pone.0060836

Date of Submission

November 2014

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.

Rights

Copyright: © 2013 Costa et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Is Part Of

VCU Physiology and Biophysics Publications