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.
Highlights
Planar lipid bilayers (PLB) have been used to study the electrophysiological aspects of many types of ion channels since the early 1960’s [1,2,3,4,5,6]
We demonstrate that a comprehensive lipid bilayer method we have termed wicking can successfully reconstitute the chloride selective channel CLIC1, and the homomultimeric protein nanopore a-hemolysin
High-resolution crystal structure of soluble CLIC1 does not show a protein with any type of pore or channel-like components, which suggests that dramatic conformational rearrangements are required for integration into the membrane [34,35]
Summary
Planar lipid bilayers (PLB) have been used to study the electrophysiological aspects of many types of ion channels since the early 1960’s [1,2,3,4,5,6]. Once a stable bilayer is achieved, the investigator frequently faces the further difficulty of long, unpredictable time intervals required for observation of proteoliposome fusion. This unpredictable parameter is often the rate-limiting step in a successful PLB experiment. Many approaches have been developed in an effort to mitigate this variable, including introducing osmoticants in the buffer such as glycerol or urea. These can increase the rate of fusion, and add chemical complexity to the system [7,8]. Despite the limitations mentioned above, the PLB technique when properly executed is arguably the most powerful method for studying the biophysics of single ion channels in a controlled environment
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