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.

Rapid lipid bilayer membrane formation on Parylene coated apertures to perform ion channel analyses

“…The inserts show the corresponding current signals after LBL formation dissolved in organic solvent, is in the range of 4.2 to 13 fF/µm 2 . Results obtained from following parallel LBL formation experiments demonstrated consistent C s compared to the values found in the literature (Kramar et al 2010;Costa et al 2013;.…”

Shaped Apertures in Photoresist Films Enhance the Lifetime and Mechanical Stability of Suspended Lipid Bilayers

“…As a reference, we first evaluated bilayer stability in the cylindrical SU8 aperture with a diameter of 60 mm. These bilayers were stable for up to 2 h and disintegrated at potentials >j150 mVj (n ¼ 8), which is similar to bilayers suspended in a conventional aperture in a Teflon septum (53,54). In marked contrast, painted bilayers in the beak-shaped and triangleshaped apertures with a tip thickness of~2 mm were stable for >20 h (n ¼ 6) at applied potentials of >j100 mVj, continuously showing alamethicin channel activity ( Fig.…”

Organelle membrane derived patches: reshaping classical methods for new targets

“… 11 ). Although this has not become a universal trouble-free approach, it prompted more recent attempts at perfecting the protein-insertion protocols, such as 12 .…”