Lipid Bilayer Experiments with Contact Bubble Bilayers for Patch-Clampers

Summary

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.

Abstract

Lipid bilayers provide a unique experimental platform for functional studies of ion channels, allowing the examination of channel-membrane interactions under various membrane lipid compositions. Among them, the droplet interface bilayer has gained popularity; however, the large membrane size hinders the recording of low electrical background noise. We have established a contact bubble bilayer (CBB) method that combines the benefits of planar lipid bilayer and patch-clamp methods, such as the ability to vary the lipid composition and to manipulate the bilayer mechanics, respectively. Using the setup for conventional patch-clamp experiments, CBB-based experiments can be readily performed. In brief, an electrolyte solution in a glass pipette is blown into an organic solvent phase (hexadecane), and the pipette pressure is maintained to obtain a stable bubble size. The bubble is spontaneously lined with a lipid monolayer (pure lipids or mixed lipids), which is provided from liposomes in the bubbles. Next, the two monolayer-lined bubbles (~50 µm in diameter) at the tip of the glass pipettes are docked for bilayer formation. Introduction of channel-reconstituted liposomes into the bubble leads to the incorporation of channels in the bilayer, allowing for single-channel current recording with a signal-to-noise ratio comparable to that of patch-clamp recordings. CBBs with an asymmetric lipid composition are readily formed. The CBB is renewed repeatedly by blowing out the previous bubbles and forming new ones. Various chemical and physical perturbations (e.g., membrane perfusion and bilayer tension) can be imposed on the CBBs. Herein, we present the basic procedure for CBB formation.

Introduction

For ion channels, the cell membrane is not simply a supporting material but a partner for generating the ion flux. Functionally, the membrane is an electrical insulator in which ion channels are embedded, and all cell membranes are imparted with a resting membrane potential. Conventionally, an arbitrary membrane potential was imposed from an external circuit by which electrical current through the channels was measured. This quantitative evaluation of the ion flux at different membrane potentials revealed the molecular properties of these channels, such as their ion-selective permeation and gating functions^1,2....

Protocol

1. Prepare Liposomes

1. Disperse phospholipids (e.g., 10 mg in powder) in chloroform at a desired concentration (e.g., 10 mg/mL).
2. Evaporate chloroform.
   1. Place the phospholipid solution in a round-bottom flask and set it on a rotary evaporator (see Table of Materials) connected to a N₂ gas cylinder. Rotate the flask under N₂ flow at room temperature until a thin phospholipid film appears (after ~30 min).
   2. Place the open flask into a desiccator that is connected to a vacuum pump. Using the vacuum pump, aspirate the inside of the desiccator for several hours to remove the chl....

Results

A typical CBB had a diameter of 50 µm (Figure 5, 6) and the specific membrane capacitance in hexadecane was 0.65 µF/cm². The bubble size was arbitrarily controlled by the intra-bubble pressure. When small bubbles are necessary for low-noise recordings, the tip diameter should be correspondingly small. For example, for a bubble size of 50 µm in diameter, the tip diameter should be 30 µm.

Discussion

The CBB method of lipid bilayer formation is based on the principle of a water-in-oil droplet lined by a monolayer²⁰. Technically, the procedures for forming CBBs are easy, especially for patch-clamp researchers, who are proficient in manipulating glass micropipettes. The electrophysiological setup for the patch clamp is readily used in the CBB when two pipette manipulators with microinjectors are available. On the other hand, because the CBB is a successor of the conventional PLB, for which a lar.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Azolectin (L-α-Phosphatidylcholine, Type IV-S)	Sigma-Aldrich	P3644
A/D Converter	Molecular Divices	Digidata1550A
Ag/AgCl electrode	Warner Instruments	64-1317
Bath Sonicator	Branson	M1800H-J
Camera	Hamamatsu Photonics	C11440-10C
Glass Capillary	Harvard Apparatus	30-0062
Hepes	Dojindo	342-01375
Hole Slideglass	Matsunami Glass	S339929
Inverted Microscope	Olympus	IX73
Isolation Table	Herz	TDI-86LA(Y)2
Micro Injenctor	Narishige	IM-11-2
Micro Manipulator	Narishige	EMM
Microforge	Narishige	MF-830
Micropipette holder
n-Hexadecane	Nacalai	07819-32
Patch-Clamp Amplifier	HEKA	EPC800
Pipette Puller	Sutter Instrument Co.	P-87
POPC (1-palmitoyl-2-oleoyl-glycero-3-phosphocholine)	Avanti Polar Lipids	850457
POPE (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine )	Avanti Polar Lipids	850757
POPG (1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) )	Avanti Polar Lipids	840457
Potassium Chloride	Nacalai	28514-75
Rotary Evapolator	Iwaki	REN-1000
Succinic Acid	Nacalai	32402-05
Vacuum Pump	Buchi	V-100