Lipid Bilayer Experiments with Contact Bubble Bilayers for Patch-Clampers

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. 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 bilayers^3,4, 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 PLB^25,26, tip-dip^22,23,27, and patch-clamp^28,29,30,31 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) method^20,32. 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 difference^34,35. 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 technique^36,37 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 = Γ_i dµ_i, where Γ_i is the surface excess of compound i, and µ_i 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: γ_bi = γ_mo cos(θ), where γ_bi is the bilayer tension, γ_mo 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: γ_mo = C_m V²/4 (cos(θ₀) – cos(θ_v)), where C_m is the membrane capacitance, V is the membrane potential, and θ₀ and θ_v 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 monolayers^43,44. 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 effect^46,47. On the other hand, flip-flops across the monolayers are slow^48,49,50.

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 membranes^15,32. 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.