Wicking: a rapid method for manually inserting ion channels into planar lipid bilayers.

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

Electron micrographs of proteoliposomes prepared under different conditions.

<p>Scale bars represent 200 nm. <b>A.</b> Bath sonication for 3 minutes at 80 kHz generated fairly uniform vesicle sizes that averaged 63.4±6.5 nm in diameter. <b>B.</b> Lipid extrusion through a polycarbonate filter formed vesicles ranging from 10–50 nm in size with an average diameter of 38.8.±4.2 nm <b>C.</b> Bath sonication for only 90 seconds generated a wide range of vesicle sizes ranging from 20–110 nm with a mean diameter of 68.7±14.4 nm.</p

A schematic for the wicking stroke superimposed on an actual image of the aperture with the lipid membrane seen through the microscope.

<p>The glass brush coated in proteoliposomes touches down in the general location of the green circle, and initiates the wicking maneuver that ends roughly where the red circle is. The black arrow represents a constant vector for the brush across the two points. Blue arrow shows the clockwise rotation of the brush, which in actual practice is roughly 100 degrees between the two points.</p

CLIC1 maintains chloride selectivity after reconstitution by wicking.

<p>4A shows single channel current traces obtained at different holding potentials under symmetric concentrations of KCl (140 mM). Traces were later converted into amplitude histograms to calculate current and then plotted against the test voltages. Applying a linear fit to the current/voltage relationship gave a single channel slope conductance of 30.8 pS for this individual experiment (4B). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060836#pone-0060836-g004" target="_blank">Figure 4C</a> summarizes six of these experiments carried out under symmetrical concentrations of KCl. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060836#pone-0060836-g004" target="_blank">Figure 4D</a> summarizes experiments conducted under a seven-fold KCl gradient and shows a shift in the reversal potential of −46 mV.</p

Modified box plots of Type A blockade events using oligonucleotides of different lengths and compositions.

<p>Box whiskers represent the maximum and minimum of the statistically significant events in the data sets. Lower and upper bounds of the boxes represent the first and third interquartile values of the data sets. Line within the box is the median of the data. Asterisks are outliers in the data set defined as 3× the Interquartile Range. Degree signs represent suspected outliers defined as 1.5× the Interquartile Range.</p

Reconstitution of CLIC1 using the wicking technique.

<p><b>A</b>) A thirty-second recording from a lipid bilayer film with a unilamellar thickness that has been verified using the membrane capacitance test. No protein has been wicked onto this bilayer. <b>B</b>) A thirty-second recording of the same bilayer as in A, after wicking mock proteoliposomes, which lack any protein. <b>C</b>) A thirty-second recording of the same bilayer, wicked with proteoliposomes containing the purified protein CLIC1. Inset shows a magnified portion of the CLIC1 trace exhibiting clear sub-conductive states. <b>D</b>) A thirty-second recording of the same bilayer following addition of 50 <b>μ</b>M IAA94 on the cis side of the bilayer. All recordings were collected at the indicated holding potential and open probability is reported for each individual trace.</p