Neuroscience
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Lipid Bilayer Experiments with Contact Bubble Bilayers for Patch-Clampers
Chapters
Summary January 16th, 2019
Here, we present a protocol for the formation of lipid bilayers using a contact bubble bilayer method. A water bubble is blown into an organic solvent, whereby a monolayer is formed at the water-oil interface. Two pipettes are manipulated to dock the bubbles to form a bilayer.
Transcript
The Contact Bubble Bilayer method is an alternative technique for conventional Lipid Bilayers. and this method allows more versatile vibration through the membrane. The CBB method integrates the benefits of Planar Lipid Bilayer and patch clamp method.
As a lipid bilayer technique a CBB with an arbitrary lipid composition can be formed. We have developed a CBB for single channel current recordings with high signal to noise ratio. This method allows varieties of membrane vibrations and offers a vast type platform to the channel membrane interface.
Everybody has experienced blowing a soap bubble. Similar maneuvers are taken for CBB formation by manually fine-tuning the applied pressure rather than blowing by mouth. Practice and enjoy the CBB.
To being this procedure disperse phospholipids in chloroform at a desired concentration in a round bottom flask. Place the phospholipid solution on a rotary evaporator connected to a nitrogen gas cylinder. Rotate the flask under nitrogen flow at room temperature until a thin phospholipid film appears.
Next place the open flask into a desiccator that is connected to a vacuum pump. Using the vacuum pump aspirate the the inside of the desiccator for several hours to remove the chloroform thoroughly. Subsequently add an appropriate volume of electrolyte solution to the flask and suspend the phospholipid to obtain a two milligrmas per milliliter phospholipid suspension.
Sonciate the suspension for 20 to 30 seconds using a bath sonicator to obtain a multilayered vesicle suspension. For the preparation of proteoliposomes that contain ion channel proteins, add a protein solution to the multilayered vesicle suspension. And sonicate for several seconds using the bath sonicator.
To prepare large bore glass pipettes, place a glass capillary in a pipette puller and fabricate the micropipette with a fine tapered tip through two step pulling. Then set the micropipette on a micro forge and contact the tip of the micropipette to a platinum filament at the tapered portion with a diameter of 30 to 50 micrometers. Heat the filament briefly and immediately turn it off.
Using distilled water and ethanol clean the surface of the glass slide with a shallow well. Next apply the siliconizing reagent on to the glass slide and let the reagent dry completely in the air. Then place the glass slide on the stage of an inverted microscope.
Add 100 microliters of hexadecane into the shallow well of the siliconized glass slide. Using a tuberculin syringe fill the electrolyte solution up to half the length of the micropipette. Next set the micropipette onto the micropipette holder with the pressure port, allowing the silver/silver chloride wire electrode to soak in the pipette electrolyte solution.
Connect one of the micropipette holders to the head stage of a patch clamp amplifier and the other one to the electrical ground. Subsequently connect a microinjector to the pressure port of the micropipette holder. Adjust the micropipette to an appropriate position above the stage of an inverted microscope using the micromanipulator.
To adjust the electrode offset potential, place one microliter of the same electrolyte solution used to fill the micropipette on the flat surface around the shallow well of the glass slide to create an electrolyte dome. Soak the tip of both micropipettes in the electrolyte dome. Afterward adjust the electrode offset potential of the patch clamp amplifier.
To draw the liposome solution from the tip, add one microliter of liposome solution on the flat surface around the shallow well of the slide glass. Next place the tip of the micropipette in the liposome containing dome. Aspirate the liposome containing solution using the microinjector.
Repeat the procedure for the other mircopipette for aspirating channel reconstituted liposomes. Now place the of the micropipette in the hexadecane in the shallow well. Blow a water bubble slowly by increasing the pressure until the bubble reaches the desired size and maintain the same pressure thereafter.
Discard the bubble by passing the tip through the oil-air interface if it is hard to keep the size of the bubble stable. Repeat the procedures until two stable bubbles are formed. Establish a contact between the two bubbles.
Fine-tune the pressure to maintain the bubble size, because the size may gradually change even at the constant intra-bubble pressure. Set the membrane potential to the appropriate value using the patch claim amplifier and wait for the channel current to emerge. For stable lipid bilayer formation it is essential to clean the blotters.
Glass capillary or pipette preparation should be washed thoroughly and avoid contamination by the detergent molecules. Once the CBB had formed the channel molecules either in aqueous solution or in the liposome were spontaneously inserted into the bilayer within the span of a few to dozens of minutes. Insertions of the channels was confirmed by the step wise increase of current amplitude under the applied membrane potential.
A bilayer formed by the CBB method can be disintegrated into two monolayers. The pTB channel current emerged immediately after attaching the two monolayers. And the current became larger as the are of contact increased the amplitude of the current was synchronized with the detach attach manipulation of the CBB.
If the number blow outs of bubbles increases the lipid concentration in the aqueous solution decreases and the monolayer is not formed. Then try to relieve pressure on liposome section. Once the CBB formation and channel reconstitution are established you can try to vary compositions of aqueous solution and membrane via profusion or injecting a hydrophilic or hydrophobic substances.
The CBB method has opened versatile ways to explore channel-membrane interplays.
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