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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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 functions1,2. The membrane platform for functional studies of ion channels is either the cell membrane or the lipid bilayer membrane. Historically, single-channel electrical current recordings were first performed in lipid bilayers3,4, and the relevant techniques were developed for cell membranes, such as the patch-clamp method (Figure 1A)5,6. Since then, these two techniques have evolved separately for different purposes (Figure 1)7,8.

Membrane lipids and bilayer membranes are currently the focus of research for their roles in supporting the structure and function of channel proteins. Therefore, the ready availability of methods to vary the lipid composition in bilayers is in high demand. Lipid bilayer formation methods such as the planar lipid bilayer (PLB)8,9,10,11, water-in-oil droplet bilayer12, and droplet interface bilayer (DIB)13,14,15,16,17,18,19 techniques (Figure 1) are common choices, providing an opportunity for examining the channel function under varying lipid compositions20. Although the DIB is technically much easier to produce than the conventional PLB, the large size of the DIB has created a disincentive for patch-clampers to apply it for studying single-channel current recordings with usual-sized conductance (<100 pS).

To circumvent the background noise, the bilayer area must be minimized. This issue recalls the repetitions of history in developing electrophysiological techniques for lipid bilayers (Figure 1). In the early days, a small-sized bilayer (1-30 µm in diameter) was formed at the tip of a pipette (tip-dip method; Figure 1C)21,22,23, rather than using a free-standing bilayer (~100 µm in diameter) on a hydrophobic septum in a chamber (Figure 1B). The tip-dip method allowed for electrical measurements with much lower background noise24. Our experiences with the PLB25,26, tip-dip22,23,27, and patch-clamp28,29,30,31 methods led us to a novel idea of forming lipid bilayers by using the principles of the water-in-oil bilayer. We have referred to this as the contact bubble bilayer (CBB) method20,32. In this method, rather than hanging the water droplets in an oil phase (Figure 1D), a water bubble is blown from a glass pipette (with tip diameter of approximately 30 µm) into the oil phase (Figure 1E and 2), where the bubble is maintained by applying a steady pressure. A monolayer forms spontaneously at the water-oil interface at the surface of the bubble. Then, two bubbles are docked through the manipulation of two glass pipettes, and the bilayer is formed as the two monolayers approach each other, yielding an equilibrium bilayer area. The size of the bubble is controlled by the intra-bubble pressure (holding pressure), and likewise the bilayer size. An average diameter of 50 µm is frequently used. Although the volume of the bubble is small (<100 pL), it is connected to the larger volume of the pipette solution that is in the microliter range, constituting the bulk electrolyte phase.

There are many benefits to use the CBB method (Table 1). As a lipid bilayer formation technique, membranes of various lipid compositions can be produced, and asymmetric membranes are more readily formed32 than are those by the conventional folding method33. The bilayer can be mechanically manipulated, unlike the conventional PLB that can only be bent with a hydrostatic pressure difference34,35. By changing the holding pressure, the bubbles either expand or shrink, leading to increased or decreased membrane tension32. The bilayer is mechanically detachable into monolayers, similar to the freeze-fracture technique36,37 of membranes in morphological studies, but with the CBB, a maneuver allows for repeated detach and attach cycles32. The small volume of the electrolyte solution within the bubble allows efficient fusion of channel-reconstituted liposomes into the bilayer, and the probability of getting channel recordings is much higher than with the conventional PLB technique. The small bubble volume also allows rapid perfusion (within ~20 ms) once another injection pipette is inserted into either of the bubbles. Unlike the patch-clamp method, once broken, a CBB membrane is re-formed immediately and repeatedly, and pipettes can be used several times a day. By integrating benefits of the patch-clamp and PLB methods, the CBB provides a versatile platform to vary the physicochemical conditions of the membrane, allowing for unprecedented studies of channel-membrane interactions.

Before presenting a detailed protocol of the CBB formation process, the physicochemical background of the bilayer formation is presented first,which will be helpful for patch-clampers to resolve experimental difficulties relating to the membrane formation that are encountered.

CBB experiments impart lessons of surface chemistry science38. The CBB is similar to a soap bubble blown from a straw into the air, where likewise, a water bubble is blown into an organic solvent. One will notice that a water bubble is hardly inflated when membrane lipids are not included in either the water bubble or the organic solvent. In the absence of amphipathic lipids, the surface tension at a water-oil interface is high, and the intra-bubble pressure for blowing a bubble will be high. This is a realization of the Laplace equation (ΔP = 2 γ/R, where ΔP is the intra-bubble pressure, γ is the surface tension, and R is the bubble radius). When the concentration of lipids in either the organic phase or the electrolyte solution is high, the density of lipids in the monolayer increases, as dictated by the Gibbs adsorption isotherm (-dγ = Γi dµi, where Γi is the surface excess of compound i, and µi is the chemical potential of component i)39, leading to a lower surface tension and ease of bubble formation. In the CBB, the bilayer can be observed from a tangential angle (Figure 2), and the contact angle between the monolayer and bilayer is measurable. This angle represents an equilibrium between the surface tensions of the monolayer and bilayer (Young equation: γbi = γmo cos(θ), where γbi is the bilayer tension, γmo is the monolayer tension, and θ is the contact angle). The changes in the contact angle indicate changes in the bilayer tension, given that the monolayer tension is evaluated from changes in the contact angle as a function of the membrane potential (Young-Lippmann equation: γmo = Cm V2/4 (cos(θ0) - cos(θv)), where Cm is the membrane capacitance, V is the membrane potential, and θ0 and θv are the contact angles at 0 and V mV, respectively)40,41,42. When two bubbles are close enough, they approach each other spontaneously. This is due to the van der Waals force, and we can visually observe this dynamic process in CBB formation.

A CBB system consists of distinct phases: namely, a bulk oil phase, water bubbles coated with a monolayer, and a contacting bilayer (Figure 3). These are reminiscent of the multiple phases observed in a PLB, such as a solvent-containing torus around the bilayer phase and a thin organic phase sandwiched by two monolayers43,44. In the CBB, the monolayer phase is continuous with the bilayer leaflet, and lipid molecules readily diffuse between the monolayer and the leaflet. The monolayer phase covers most of the bubble surface, constituting the major phase that serves as a lipid reservoir. Because the hydrophobic tail of lipids in the monolayer extends outward to the bulk oil phase, the bilayer interior or the hydrophobic core opens to the bulk oil phase. Thus, a hydrophobic substance injected into the oil phase close to the bilayer is able to readily access the bilayer interior. This is the membrane perfusion technique we had developed recently45, by which the lipid composition in the bilayer is changed rapidly (within a second) during single-channel current recordings. We found that the cholesterol content in the bilayer could be reversibly controlled by switching the cholesterol perfusion on and off45. In the event that the concentration of the relevant substance in the monolayer and bilayer differs, the concentration gradient of the relevant substance is immediately dissolved through diffusion, which is known as the Marangoni effect46,47. On the other hand, flip-flops across the monolayers are slow48,49,50.

Using the CBB method, the bilayer is formed under versatile physicochemical conditions, such as an electrolyte pH as low as 1 51, a salt (K+, Na+, etc.) concentration up to 3 M, a membrane potential as high as ±400 mV, and a system temperature of up to 60 °C.

There are several options for formation of the CBB and incorporation of channel molecules therein. For formation of the monolayer at the water-oil interface, lipids are added either in an organic solvent (lipid-out method; Figure 4A, 4C) or in a bubble as liposomes (lipid-in method; Figure 4B, 4D). Notably, the lipid-in method allows for the formation of asymmetric membranes15,32. Channel molecules soluble in aqueous solution (e.g., channel-forming peptides) are directly added into the bubble (Figure 4A, B)52,53, whereas channel proteins are reconstituted into liposomes, which are then added into the bubble (Figure 4C, D). Herein, the formation of CBBs by the lipid-in method for either a channel peptide (polytheonamide B (pTB); Figure 4A) or a protein (KcsA potassium channel, Figure 4C) is shown.

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 chloroform thoroughly.
  3. Add an appropriate volume of an electrolyte solution to the flask and suspend the phospholipid to obtain a 2 mg/mL phospholipid suspension.
  4. Sonicate the suspension for several tens of seconds using a bath sonicator (see Table of Materials) to obtain a multilayered vesicle (MLV) suspension.
  5. For preparation of proteoliposomes that contain ion channel proteins, add a protein solution (with the proteins solubilized using appropriate detergents; 2% volume is the maximum) to the MLV suspension and sonicate for several seconds using the bath sonicator.

2. Prepare Large-Bore Glass Pipettes

  1. Set a glass capillary to a pipette puller and fabricate micropipettes with a fine tapered tip through two-step pulling.
  2. Set the micropipette on a microforge and contact the tip of the micropipette to a platinum filament at the tapered portion with a diameter of 30 to 50 µm.
  3. Heat the filament briefly (5 s) and immediately turn it off.
    NOTE: This manipulation forms a crack at the heating point, whereby the tip of the micropipette is cut off, leaving a wide bore with a diameter of 30 to 50 µm.

3. Treat the Surface of Glass Slide with a Shallow Concave Well (Siliconization for a Water-Repellent Finish)

  1. Clean the surface of the glass slide with a shallow well with distilled water and ethanol.
  2. Apply an appropriate volume (e.g., 100 µL) of a siliconizing reagent (water-repellent) onto the hole slide glass.
  3. Dry the reagent completely in the air.
  4. Place the glass slide on the stage of an inverted microscope.

4. Form the CBB and Perform Electrophysiological Measurement

  1. Add 100 µL of hexadecane into the shallow well of the siliconized hole slide glass.
    ​NOTE: For the lipid-out method, phospholipids are dispersed in hexadecane (20 mg/mL) beforehand.
  2. Fill the electrolyte solution up to half the length of the micropipette, using a tuberculin syringe.
  3. Set the micropipette onto the micropipette holder with a pressure port, allowing the Ag/AgCl wire electrode to soak into the pipette electrolyte solution.
  4. Connect one of the micropipette holders to the head stage of a patch-clamp amplifier and the other one to the electrical ground.
  5. Connect a microinjector to the pressure port of the micropipette holder.
  6. Set the micropipette to an appropriate position above the stage of an inverted microscope by manipulating the micromanipulator.
  7. Adjust the electrode offset potential.
    1. Place 1 µL of the same electrolyte solution used to fill the micropipette on the flat surface around the shallow well of the hole slide glass, creating an electrolyte dome.
    2. Soak the tip of both micropipettes into the electrolyte dome by manipulating the micromanipulator.
    3. Adjust the electrode offset potential of the patch-clamp amplifier.
    4. Confirm the correct offset at the end of experiments by breaking the CBB via application of a high membrane potential (electrical breakdown; using Zap on the amplifier), causing the two bubbles to be fused into one (bubble fusion).
    5. Correct the liquid junction potential54 in the cases where asymmetric electrolyte solutions are used, such that the calculated value is added to the applied membrane potential for the true membrane potential.
      NOTE: The liquid junction potential is calculated using the program JPCalc55.
  8. Draw the liposome solution from the tip.
    ​NOTE: When water-soluble channels are examined using the lipid-out method, suctioning of the liposome solution is not necessary.
    1. Place 1 µL of liposome solution on the flat surface around the shallow well of the hole slide glass (liposome-containing dome).
    2. Manipulate the micromanipulator and insert the tip of the micropipette into the liposome-containing dome.
    3. Aspirate the liposome-containing solution by lowering the pressure inside the micropipette holder using the microinjector.
    4. Repeat the procedure for the other pipette.
  9. Manipulate the micromanipulator and dip the tip of the micropipette into the hexadecane in the shallow well.
  10. Blow a water bubble slowly by increasing the pressure until the bubble reaches the desired size (e.g., 50 µm in diameter) and maintain the same pressure thereafter.
  11. Discard the bubbles by passing the tip through the oil-air interface if it is hard to keep the size of the bubbles stable.
  12. Repeat steps 4.8 to 4.9 until stable bubbles are formed.
  13. Manipulate the bubbles to allow contact between them (Figure 5).
    NOTE: Sometimes, the bubbles approach each other spontaneously to form the CBB. In other cases, the bubbles are close but do not contact each other. In this case, push the bubbles each other mechanically.
  14. Fine-tune the pressure to maintain the bubble size, because the size may gradually change even at the constant intra-bubble pressure.
  15. Set the membrane potential to the appropriate value using the patch-clamp amplifier and wait for the channel current to emerge (Figure 6).

5. Measure Bilayer Capacitance

  1. Measure the bilayer electrical capacitance (Cel) by applying a ramp potential.
    NOTE: When the rate of the voltage change in the ramp command is 10 mV/10 ms (or 1 V/s) followed by -10 mV/10 ms, the amplitude of the current jump upon changes in the slope corresponds to the read of the membrane'scapacitance value (e.g., 100 pA → 100 pF).
  2. Evaluate the bilayer area of two bubbles stacked one on the other and focus the microscope at the bilayer level to view the edge of the bilayer (Figure 6).
    NOTE: The bilayer shape is mostly circular, and the area is calculated from the radius.
  3. Calculate the specific membrane capacitance (Csp) by dividing the electrical capacitance by the bilayer area (Csp = Cel/A).
  4. Calculate the bilayer thickness (thickness of the hydrophobic core) using Dc = (εrε0)/Csp (where εr and ε0 represent the permittivity of the hydrophobic region of the bilayer and the permittivity of a vacuum, respectively).

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...

Disclosures

The authors have no conflict of interest to disclose.

Acknowledgements

The authors would like to thank Mariko Yamatake and Masako Takashima for technical assistance. This work was supported in part by KAKENHI grant numbers 16H00759 and 17H04017 (SO).

Materials

NameCompanyCatalog NumberComments
Azolectin (L-α-Phosphatidylcholine, Type IV-S)Sigma-AldrichP3644
A/D ConverterMolecular DivicesDigidata1550A
Ag/AgCl electrodeWarner Instruments64-1317
Bath SonicatorBransonM1800H-J
CameraHamamatsu PhotonicsC11440-10C
Glass CapillaryHarvard Apparatus30-0062
HepesDojindo342-01375
Hole SlideglassMatsunami GlassS339929
Inverted MicroscopeOlympusIX73
Isolation TableHerzTDI-86LA(Y)2
Micro InjenctorNarishigeIM-11-2
Micro ManipulatorNarishigeEMM
MicroforgeNarishigeMF-830
Micropipette holder
n-HexadecaneNacalai07819-32
Patch-Clamp AmplifierHEKAEPC800
Pipette PullerSutter Instrument Co.P-87
POPC (1-palmitoyl-2-oleoyl-glycero-3-phosphocholine)Avanti Polar Lipids850457
POPE (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine
)		Avanti Polar Lipids850757
POPG (1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) )Avanti Polar Lipids840457
Potassium ChlorideNacalai28514-75
Rotary EvapolatorIwakiREN-1000
Succinic AcidNacalai32402-05
Vacuum PumpBuchiV-100

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