Name: lipid bilayer experiments with contact bubble bilayers for patch-clampers
Uploaded: 2019-01-16T13:45:00.000+00:00
Duration: 7 min 18 s
Description: Watch this Scientific Journal Video about Lipid Bilayer Experiments with Contact Bubble Bilayers for Patch-Clampers at JoVE.com

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).

1. Prepare Liposomes
<ol>
	<li>Disperse phospholipids (e.g., 10 mg in powder) in chloroform at a desired concentration (e.g., 10 mg/mL).</li>
	<li>Evaporate chloroform.
	<ol>
		<li>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).</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Add an appropriate volume of an electrolyte solution to the flask and suspend the phospholipid to obtain a 2 mg/mL phospholipid suspension.</li>
	<li>Sonicate the suspension for several tens of seconds using a bath sonicator (see Table of Materials) to obtain a multilayered vesicle (MLV) suspension.</li>
	<li>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.</li>
</ol>
2. Prepare Large-Bore Glass Pipettes
<ol>
	<li>Set a glass capillary to a pipette puller and fabricate micropipettes with a fine tapered tip through two-step pulling.</li>
	<li>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 &#181;m.</li>
	<li>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 &#181;m.</li>
</ol>
3. Treat the Surface of Glass Slide with a Shallow Concave Well (Siliconization for a Water-Repellent Finish)
<ol>
	<li>Clean the surface of the glass slide with a shallow well with distilled water and ethanol.</li>
	<li>Apply an appropriate volume (e.g., 100 &#181;L) of a siliconizing reagent (water-repellent) onto the hole slide glass.</li>
	<li>Dry the reagent completely in the air.</li>
	<li>Place the glass slide on the stage of an inverted microscope.</li>
</ol>
4. Form the CBB and Perform Electrophysiological Measurement
<ol>
	<li>Add 100 &#181;L of hexadecane into the shallow well of the siliconized hole slide glass. 
	&#8203;NOTE: For the lipid-out method, phospholipids are dispersed in hexadecane (20 mg/mL) beforehand.</li>
	<li>Fill the electrolyte solution up to half the length of the micropipette, using a tuberculin syringe.</li>
	<li>Set the micropipette onto the micropipette holder with a pressure port, allowing the Ag/AgCl wire electrode to soak into the pipette electrolyte solution.</li>
	<li>Connect one of the micropipette holders to the head stage of a patch-clamp amplifier and the other one to the electrical ground.</li>
	<li>Connect a microinjector to the pressure port of the micropipette holder.</li>
	<li>Set the micropipette to an appropriate position above the stage of an inverted microscope by manipulating the micromanipulator.</li>
	<li>Adjust the electrode offset potential.
	<ol>
		<li>Place 1 &#181;L 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.</li>
		<li>Soak the tip of both micropipettes into the electrolyte dome by manipulating the micromanipulator.</li>
		<li>Adjust the electrode offset potential of the patch-clamp amplifier.</li>
		<li>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).</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Draw the liposome solution from the tip. 
	&#8203;NOTE: When water-soluble channels are examined using the lipid-out method, suctioning of the liposome solution is not necessary.
	<ol>
		<li>Place 1 &#181;L of liposome solution on the flat surface around the shallow well of the hole slide glass (liposome-containing dome).</li>
		<li>Manipulate the micromanipulator and insert the tip of the micropipette into the liposome-containing dome.</li>
		<li>Aspirate the liposome-containing solution by lowering the pressure inside the micropipette holder using the microinjector.</li>
		<li>Repeat the procedure for the other pipette.</li>
	</ol>
	</li>
	<li>Manipulate the micromanipulator and dip the tip of the micropipette into the hexadecane in the shallow well.</li>
	<li>Blow a water bubble slowly by increasing the pressure until the bubble reaches the desired size (e.g., 50 &#181;m in diameter) and maintain the same pressure thereafter.</li>
	<li>Discard the bubbles by passing the tip through the oil-air interface if it is hard to keep the size of the bubbles stable.</li>
	<li>Repeat steps 4.8 to 4.9 until stable bubbles are formed.</li>
	<li>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.</li>
	<li>Fine-tune the pressure to maintain the bubble size, because the size may gradually change even at the constant intra-bubble pressure.</li>
	<li>Set the membrane potential to the appropriate value using the patch-clamp amplifier and wait for the channel current to emerge (Figure 6).</li>
</ol>
5. Measure Bilayer Capacitance
<ol>
	<li>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&#39;scapacitance value (e.g., 100 pA &#8594; 100 pF).</li>
	<li>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.</li>
	<li>Calculate the specific membrane capacitance (Csp) by dividing the electrical capacitance by the bilayer area (Csp = Cel/A).</li>
	<li>Calculate the bilayer thickness (thickness of the hydrophobic core) using Dc = (&#949;r&#949;0)/Csp (where &#949;r and &#949;0 represent the permittivity of the hydrophobic region of the bilayer and the permittivity of a vacuum, respectively).</li>
</ol>

The authors have no conflict of interest to disclose.

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 large amount of physicochemical knowledge has been accumulated8, this background as well as knowledge of surface chemistry38 is helpful for operating and manipulating CBBs. The CBB serves a versatile platform for studying channel-membrane interplays with its ability of modifying chemical composition and physical status61. In the CBB, lipids lining the bubble can diffuse freely across the border of the monolayer phase and leaflet of the bilayer, and we have developed techniques such as the bilayer attach-detach and the membrane perfusion methods45. We have extended CBBs for in vitro channel protein synthesis, where channel synthesis was performed in the bubble, and newly synthesized channel proteins were subjected to spontaneous transfer to the bilayer under application of a membrane potential where nascent channel functions of the KcsA channel was traced from the time of initiating the transcription/translation of KcsA DNA62.
Among the steps of the protocol, maintaining the bubble size is critical. The bubbles may slowly swell or shrink spontaneously because lipid transfer to the interface is slow and the monolayer tension is prone to change. In particular, the first bubble inflated just after suction of the liposome solution (steps 4.8.3 and 4.10) is hard to maintain because the monolayer tension is lowered by the liposomes accumulated at the tip of the pipette. Discarding the initial bubbles and subsequent formation of the bubbles provide the stable CBB. Visual tracing of the bubble size and fine tuning of the pressure are necessary.
There are experimental limitations to the CBB method. Although the CBB is designed for electrophysiological measurements, the electrical resistance of the bilayer for certain lipid compositions may not be high enough (e.g., 100 G&#937;) for single-channel recordings. For example, dioleoyl phosphatidylcholine is frequently used for liposomes, but it forms electrically leaky CBBs at 25 &#176;C63. CBBs cannot even be formed with lipid species whose phase transition temperature is above the recording temperature. Indeed, CBB formation was difficult with dipalmitoyl phosphatidylcholine at room temperature, but raising the temperature above Tm circumvented the problem64. In these experiments, the temperature was controlled by using a transparent heat plate (see Table of Materials) beneath the slide glass62. The phase transition temperature of the frequently used lipid diphytanoyl phosphatidylcholine is below 0 &#176;C 65, and CBBs are readily formed32,45,51 at a broad temperature range.
Phospholipids of the water-oil interface are provided from either the lipid-in or lipid-out method, where the transfer rate is relatively slower in the lipid-out than in the lipid-in method63. In addition, for reasons unknown, the electrical resistance of the bilayer membrane is relatively smaller with the lipid-out method than with the lipid-in method.
In the protocol (step 4.8), a solution of liposomes and channel-reconstituted liposomes (1 &#181;L) is loaded from the tip of the pipette (steps 4.9 and 4.10), and the rest of the pipette contains the previously filled electrolyte solution. This is merely for conserving materials, such as lipids and channel molecules. Consequently, the liposomes diffuse gradually toward the upper pipette solution, and after a while, the lipid concentration at the tip of the pipette becomes insufficient to form lipid monolayers. In this case, fresh liposome solution should be aspirated from the tip (step 4.8). This temporal concentration change can be circumvented when the whole pipette is filled with the solution containing lipids and channel molecules, whereas liposomes gradually settle down into the bubbles. In this case, bubbles are renewed.
Electrophysiologically, the CBB size is larger than that of the patch clamp, thus yielding a larger membrane capacitance. However, the series resistance (the electrical resistance in a series of membrane resistance) is much lower (~100 k&#937;) than that for the patch clamp (pipette resistance &#62; 1 M&#937;), which accelerates the speed of the voltage clamp and attenuates the background noise and series resistance errors in the voltage-clamped membrane potentials66,67,68. The background noise is a function of the membrane capacitance and the series resistance, where a low series resistance complements a high membrane capacitance, resulting in low background noise9.
As a rule of lipid bilayer experiments, detergents should not be used to wash glassware. Even a trace amount of detergent perturbs the integrity of the bilayer. Organic solvents such as chloroform/methanol and ethanol should be used for such cleaning purposes instead.
Overall, the CBB integrates the benefits of both the patch clamp (e.g., the mechanical manipulation of the membrane) and the PLB (e.g., the ability to modify the lipid composition of the membrane). Various types of channel-forming substances and channel proteins have been studied52,53,69,70. The development of this method is opportune since increasingly more researchers are focusing on channel-membrane interplay, and the CBB provides a versatile platform for various experiments. Further experimental developments in the CBB method are expected.

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 (&#60;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 &#181;m 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 &#181;m 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 &#181;m) 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 &#181;m is frequently used. Although the volume of the bubble is small (&#60;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 (&#916;P = 2 &#947;/R, where &#916;P is the intra-bubble pressure, &#947; 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 (-d&#947; = &#915;i d&#181;i, where &#915;i is the surface excess of compound i, and &#181;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: &#947;bi = &#947;mo cos(&#952;), where &#947;bi is the bilayer tension, &#947;mo is the monolayer tension, and &#952; 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: &#947;mo = Cm V2/4 (cos(&#952;0) - cos(&#952;v)), where Cm is the membrane capacitance, V is the membrane potential, and &#952;0 and &#952;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 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 &#177;400 mV, and a system temperature of up to 60 &#176;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.

<table><tbody><tr><td>Azolectin (L-&amp;alpha;-Phosphatidylcholine, Type IV-S)</td><td>Sigma-Aldrich</td><td>P3644</td><td></td></tr><tr><td>A/D Converter</td><td>Molecular Divices</td><td>Digidata1550A</td><td></td></tr><tr><td>Ag/AgCl electrode</td><td>Warner Instruments</td><td>64-1317</td><td></td></tr><tr><td>Bath Sonicator</td><td>Branson</td><td>M1800H-J</td><td></td></tr><tr><td>Camera</td><td>Hamamatsu Photonics</td><td>C11440-10C</td><td></td></tr><tr><td>Glass Capillary</td><td>Harvard Apparatus</td><td>30-0062</td><td></td></tr><tr><td>Hepes</td><td>Dojindo</td><td>342-01375</td><td></td></tr><tr><td>Hole Slideglass</td><td>Matsunami Glass</td><td>S339929</td><td></td></tr><tr><td>Inverted Microscope</td><td>Olympus</td><td>IX73</td><td></td></tr><tr><td>Isolation Table</td><td>Herz</td><td>TDI-86LA(Y)2</td><td></td></tr><tr><td>Micro Injenctor</td><td>Narishige</td><td>IM-11-2</td><td></td></tr><tr><td>Micro Manipulator</td><td>Narishige</td><td>EMM</td><td></td></tr><tr><td>Microforge</td><td>Narishige</td><td>MF-830</td><td></td></tr><tr><td>Micropipette holder</td><td></td><td></td><td></td></tr><tr><td>n-Hexadecane</td><td>Nacalai</td><td>07819-32</td><td></td></tr><tr><td>Patch-Clamp Amplifier</td><td>HEKA</td><td>EPC800</td><td></td></tr><tr><td>Pipette Puller</td><td>Sutter Instrument Co.</td><td>P-87</td><td></td></tr><tr><td>POPC (1-palmitoyl-2-oleoyl-glycero-3-phosphocholine)</td><td>Avanti Polar Lipids</td><td>850457</td><td></td></tr><tr><td>POPE (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine&lt;br/&gt; )</td><td>Avanti Polar Lipids</td><td>850757</td><td></td></tr><tr><td>POPG (1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1&#039;-rac-glycerol) )</td><td>Avanti Polar Lipids</td><td>840457</td><td></td></tr><tr><td>Potassium Chloride</td><td>Nacalai</td><td>28514-75</td><td></td></tr><tr><td>Rotary Evapolator</td><td>Iwaki</td><td>REN-1000</td><td></td></tr><tr><td>Succinic Acid</td><td>Nacalai</td><td>32402-05</td><td></td></tr><tr><td>Vacuum Pump</td><td>Buchi</td><td>V-100</td><td></td></tr></tbody></table>

lipid bilayer experiments with contact bubble bilayers for patch-clampers

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 &#181;m 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.

A typical CBB had a diameter of 50 &#181;m (Figure 5, 6) and the specific membrane capacitance in hexadecane was 0.65 &#181;F/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 &#181;m in diameter, the tip diameter should be 30 &#181;m.
Once the CBB had formed, the channel molecules either in aqueous solution or in the liposome were spontaneously inserted into the bilayer within a span of a few to dozens of minutes. Insertion of the channels was confirmed by the stepwise increase of current amplitude (Figure 7) under the applied membrane potential. The smaller membrane area (&#60;1,000 &#181;m2) relative to that in the conventional PLB and DIB systems substantially improved the electrical signal-to-noise ratio.
Current recordings could be continued until the membrane was disrupted and the two bubbles merged. New bubbles were blown and the CBB formed immediately and repeatedly. The pipette could be used repeatedly within a day.
Duty cycle of attach and detach. A bilayer formed by the CBB method can be disintegrated into two monolayers. Detach-attach of the CBB can be repeated by manipulating the two bubbles (duty cycle of detach-attach). This process was monitored by the appearance of the channel current of pTB, a peptide channel from a marine sponge, as it was readily inserted into the lipid bilayer to form a monovalent cation-selective pore52,56. The pTB channel current emerged immediately after attaching the two monolayers, and the current became larger as the area of contact increased (Figure 8). The amplitude of the current was synchronized with the detach-attach manipulation of the CBB.
Single-channel measurements of the KcsA channel. The KcsA potassium channel is pH sensitive, being activated by acidic intracellular pH57. Thus, the electrolyte solution was set to be asymmetric58,59,60. In step 4.2 of CBB formation, the pipette solution of the left side was set at pH 4, whereas that of the right side was set at pH 7.525. In step 4.7.1, a proteoliposome suspension, rather than liposome suspension, was placed onto the slide glass for aspiration into the left pipette. Accordingly, the KcsA channel was oriented in the membrane with its cytoplasmic domain facing the left bubble. Liposomes with the protein:lipid weight ratio of 1:2000 are suitable for single-channel current recording, and those with a 1:10 ratio are for macroscopic current recording.
Asymmetric membrane. An asymmetric lipid bilayer can be formed using different liposome suspensions for each bubble (lipid-in)15,32. The orientation of the KcsA channel in the membrane was regulated by setting an asymmetric solution pH (pH 4in/pH 7out), leading to its cytoplasmic side being toward the left side. Accordingly, the left side was assigned as &#34;in,&#34; whereas the right side was assigned as &#34;out.&#34; To examine lipid dependence on the gating of the KcsA channel, four types of CBB (including an asymmetric CBB) were used; namely, PGin/PGout, PGin/PCout, PCin/PGout, and PCin/PCout (PG: phosphatidylglycerol) (Figure 9). The KcsA channel exhibited high open probability (&#62;90%) only in the PGin/PGout and PGin/PCout membranes. The KcsA channel requires the existence of anionic phospholipids in the inner leaflet of the membrane for a high open probability26.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58840/58840fig1.jpg" /> 
Figure 1:&#160;Lipid bilayer and patch-clamp methods. Various methods have been developed for forming the lipid bilayer. (A) Patch-clamp method. (B) Conventional planar lipid bilayer method, which provides a free-standing, mostly vertical, bilayer on a small hole. (C) Tip-dip method. A monolayer at the air-water interface is positioned on the tip of a glass electrode. Various modifications have been developed. (D) Droplet interface bilayer method. (E) Contact bubble bilayer method. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/58840/58840fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58840/58840fig2.jpg" /> 
Figure 2:&#160;Contact bubble bilayer formation. As a bubble is blown into an oil phase, lipid molecules transfer spontaneously to the water-oil interface. Different types of lipids as liposomes are included in each bubble (lipid-in), and monolayers are formed exclusively by the relevant lipids. Docking two monolayers generates an asymmetric bilayer membrane. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/58840/58840fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58840/58840fig3.jpg" /> 
Figure 3:&#160;Distinct phases in the contact bubble bilayer, and the dynamics of lipids therein. Lipids lining the bubble constitute a phase where they belong to either a monolayer or a bilayer. Flip-flop of lipids across the bilayer is infrequent, and the asymmetric membrane is retained for a long time. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/58840/58840fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58840/58840fig4.jpg" /> 
Figure 4:&#160;Delivering lipids with the lipid-out or lipid-in method. Lipids are added either in an organic solvent (lipid-out; A and C) or in aqueous solution as liposomes (lipid-in; B and D). In the lipid-in method, an asymmetric membrane is formed. Channel-forming substances that are soluble in aqueous solution (blue), such as peptides, are added into one of the bubbles and spontaneously inserted into the bilayer. In the lipid-in method, a part of the channels is inserted into liposomes, which are then fused to the bilayer. Channel proteins (red) are reconstituted into liposomes in either the lipid-out or lipid-in method, which are then added into one of the bubbles and spontaneously fused to the bilayer. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/58840/58840fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/58840/58840fig5.jpg" /> 
Figure 5:&#160;Formation and microscopic image of the contact bubble bilayer. The bilayer is observed from a tangential direction. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/58840/58840fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/58840/58840fig6v2.jpg" /> 
Figure 6:&#160;Observation of the bilayer for measuring the bilayer area. Two bubbles are stacked one on the other through pipette manipulation, and the microscope is focused at the level of the contact bubble bilayer. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/58840/58840fig6v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/58840/58840fig7v2.jpg" /> 
Figure 7:&#160;Stepwise insertion of the KcsA channel into the contact bubble bilayer membrane. The E71A mutant of the KcsA channel was used to show the immediate detection of the inserted channels. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/58840/58840fig7v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/58840/58840fig8.jpg" /> 
Figure 8:&#160;Detach-attach of monolayers and responses of channels. A peptide channel, polytheonamide B, was added into one of the bubbles, which was then spontaneously incorporated into the membrane. When detached, channels in the membrane withdrew but were retained at the membrane leaflet of the channel-added side. Upon attachment, the channels were inserted into the membrane. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/58840/58840fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/58840/58840fig9.jpg" /> 
Figure 9:&#160;Formation of an asymmetric membrane and channel activity dependent on the composition of the leaflet. The KcsA channel is regulated by acidic lipids, such as PG, in the inner leaflet of the membrane. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/58840/58840fig9large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td></td>
			<td>Conventional PLB</td>
			<td>Liposome patch</td>
			<td>DIB</td>
			<td>CBB</td>
		</tr>
		<tr>
			<td>Success rate1</td>
			<td>high</td>
			<td>low</td>
			<td>very high</td>
			<td>very high</td>
		</tr>
		<tr>
			<td>Immediate reformability2</td>
			<td>yes</td>
			<td>no</td>
			<td>intermediate</td>
			<td>yes</td>
		</tr>
		<tr>
			<td>Asymmetric membrane</td>
			<td>yes</td>
			<td>no</td>
			<td>yes</td>
			<td>yes</td>
		</tr>
		<tr>
			<td>Solution exchange</td>
			<td>slow</td>
			<td>very fast</td>
			<td>slow</td>
			<td>fast</td>
		</tr>
		<tr>
			<td>S/N ratio3</td>
			<td>low</td>
			<td>high</td>
			<td>low</td>
			<td>high</td>
		</tr>
		<tr>
			<td>Membrane deformation</td>
			<td>no</td>
			<td>no</td>
			<td>no</td>
			<td>yes4</td>
		</tr>
		<tr>
			<td>Mechanical manipulation</td>
			<td>no</td>
			<td>yes5</td>
			<td>yes</td>
			<td>yes</td>
		</tr>
		<tr>
			<td>Membrane perfusion</td>
			<td>no</td>
			<td>no</td>
			<td>yes</td>
			<td>yes</td>
		</tr>
		<tr>
			<td>Lipid compositions</td>
			<td>diverse</td>
			<td>limited6</td>
			<td>diverse</td>
			<td>diverse</td>
		</tr>
		<tr>
			<td colspan="5">1The success rate of forming stable lipid bilayer per trial.</td>
		</tr>
		<tr>
			<td colspan="5">2Formation of a new membrane quickly after break down of a membrane. Also, membrane is formed repeatedly.</td>
		</tr>
		<tr>
			<td colspan="5">3The signal-to-noise (S/N) ratio represents the electrical background noise primary generated from the membrane capacitance.</td>
		</tr>
		<tr>
			<td colspan="5">4Concave or convex membrane can be formed by applying different inner pressure between two bubbles.</td>
		</tr>
		<tr>
			<td colspan="5">5Only increasing the membrane tension.</td>
		</tr>
		<tr>
			<td colspan="5">6Phospholipids that can form giant unilamellar vesicles are applicable, but the giga-seal may not be attained.</td>
		</tr>
	</tbody>
</table>
Table 1: Characteristics of various lipid bilayer membranes.

Watch this Scientific Journal Video about Lipid Bilayer Experiments with Contact Bubble Bilayers for Patch-Clampers at JoVE.com

Lipid Bilayer Experiments with Contact Bubble Bilayers for Patch-Clampers

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.

Lipid bilayers provide a unique experimental platform for functional studies of ion channels, allowing the examination of channel-membrane interactions ...

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Channels and the Electrical Properties of Membranes

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9.5K Views.  University of Fukui Faculty of Medical Sciences. 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.

Video: Lipid Bilayer Experiments with Contact Bubble Bilayers for Patch-Clampers

Supported Planar Bilayers for the Formation of Study of Immunological Synapses and Kinapse

Supported planar bilayers are powerful tools that can be used to model the molecular interactions in an immunological synapse.   To mimic the interactions between lymphocytes and antigen presenting cells, we use Ni2+-chelating lipids to anchor recombinant cell adhesion and MHC proteins to the upper leaflet of a bilayer with poly-histidine tags. Planar bilayers are generated by preparing lipid, treating the glass surfaces where the bilayer will form, and then forming the bilayer in a specialized chamber containing a flow-cell where the lymphocytes will be added.  Then, bilayers are charged with Ni and his-tagged recombinant proteins are added.  Finally,  lymphocytes are injected into the flow cell and TIRF microscopy can be used to image synapse formation and the mechanisms that control T cell locomotion, sites of receptor sorting,  and sites of receptor degradation.

Supported planar bilayers are powerful tools that can be used to model the molecular interactions in an immunological synapse. Here, we show methods for anchoring cell adhesion proteins known to modulate synapse formation to the upper leaflet of the lipid bilyer and visualize synapse formation using TIRF microscopy.

Supported planar bilayers are powerful tools that can be used to model the molecular interactions in an immunological synapse.   To mimic the interactions ...

Biology

Single Molecule Methods for Monitoring Changes in Bilayer Elastic Properties

Membrane protein function is regulated by the cell membrane lipid composition.  This regulation is due to a combination of specific lipid-protein interactions and more general lipid bilayer-protein interactions. These interactions are particularly important in pharmacological research, as many current pharmaceuticals on the market can alter the lipid bilayer material properties, which can lead to altered membrane protein function.   The formation of gramicidin channels are dependent on conformational changes in gramicidin subunits which are in turn dependent on the properties of the lipid.  Hence the gramicidin channel  current is a reporter of altered properties of the bilayer due to certain compounds.  

Membrane protein function is regulated by the cell membrane lipid composition. This video-article details how to form a patch using bilayer patch electrodes, as well as how to use gramicidin channels as reporters of altered membrane properties.

Membrane protein function is regulated by the cell membrane lipid composition.  This regulation is due to a combination of specific lipid-protein ...

Preparation of Artificial Bilayers for Electrophysiology Experiments

Planar lipid bilayers, also called artificial lipid bilayers, allow you to study ion-conducting channels in a well-defined environment. These bilayers can be used for many different studies, such as the characterization of membrane-active peptides, the reconstitution of ion channels or investigations on how changes in lipid bilayer properties alter the function of bilayer-spanning channels.  Here, we show how to form a planar bilayer and how to isolate small patches from the bilayer, and in a second video  will also demonstrate a procedure for using gramicidin channels to determine changes in lipid bilayer elastic properties.   We also demonstrate the individual steps needed to prepare the bilayer chamber, the electrodes and how to test that the bilayer is suitable for single-channel measurements.

Planar lipid bilayers, also called artificial lipid bilayers, allow you to study ion-conducting channels in a well-defined environment. Here, we demonstrate the individual steps needed to prepare the bilayer chamber, the electrodes and how to test that the bilayer is suitable for single-channel measurements.

Planar lipid bilayers, also called artificial lipid bilayers, allow you to study ion-conducting channels in a well-defined environment. These bilayers can ...

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer (SALB) Method

In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related processes while conferring biocompatibility and biofunctionality to solid substrates. The spontaneous adsorption and rupture of phospholipid vesicles is the most commonly used method to form SLBs. However, under physiological conditions, vesicle fusion (VF) is limited to only a subset of lipid compositions and solid supports. Here, we describe a one-step general procedure called the solvent-assisted lipid bilayer (SALB) formation method in order to form SLBs which does not require vesicles. The SALB method involves the deposition of lipid molecules onto a solid surface in the presence of water-miscible organic solvents (e.g., isopropanol) and subsequent solvent-exchange with aqueous buffer solution in order to trigger SLB formation. The continuous solvent exchange step enables application of the method in a flow-through configuration suitable for monitoring bilayer formation and subsequent alterations using a wide range of surface-sensitive biosensors. The SALB method can be used to fabricate SLBs on a wide range of hydrophilic solid surfaces, including those which are intractable to vesicle fusion. In addition, it enables fabrication of SLBs composed of lipid compositions which cannot be prepared using the vesicle fusion method. Herein, we compare results obtained with the SALB and conventional vesicle fusion methods on two illustrative hydrophilic surfaces, silicon dioxide and gold. To optimize the experimental conditions for preparation of high quality bilayers prepared via the SALB method, the effect of various parameters, including the type of organic solvent in the deposition step, the rate of solvent exchange, and the lipid concentration is discussed along with troubleshooting tips. Formation of supported membranes containing high fractions of cholesterol is also demonstrated with the SALB method, highlighting the technical capabilities of the SALB technique for a wide range of membrane configurations.

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer SALB Method

We present an experimental protocol to form a supported lipid bilayer on solid substrates without using lipid vesicles. We demonstrate a one-step method to form a lipid bilayer on silicon dioxide and gold as well as supported membranes with cholesterol-enriched domain for various biological applications.

In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related ...

Bioengineering

Automated Lipid Bilayer Membrane Formation Using a Polydimethylsiloxane Thin Film

An artificial lipid bilayer, or black lipid membrane (BLM), is a powerful tool for studying ion channels and protein interactions, as well as for biosensor applications. However, conventional BLM formation techniques have several drawbacks and they often require specific expertise and laborious processes. In particular, conventional BLMs suffer from low formation success rates and inconsistent membrane formation time. Here, we demonstrate a storable and transportable BLM formation system with controlled thinning-out time and enhanced BLM formation rate by replacing conventionally used films (polytetrafluoroethylene, polyoxymethylene, polystyrene) to polydimethylsiloxane (PDMS). In this experiment, a porous-structured polymer such as PDMS thin film is used. In addition, as opposed to conventionally used solvents with low viscosity, the use of squalene permitted a controlled thinning-out time via slow solvent absorption by PDMS, prolonging membrane lifetime. In addition, by using a mixture of squalene and hexadecane, the freezing point of the lipid solution was increased (~16 &#176;C), in addition, membrane precursors were produced that can be indefinitely stored and readily transported. These membrane precursors have reduced BLM formation time of &#60; 1 hr and achieved a BLM formation rate of ~80%. Moreover, ion channel experiments with gramicidin A demonstrated the feasibility of the membrane system.

We demonstrate a storable, transportable lipid bilayer formation system. A lipid bilayer membrane can be formed within 1 hr with over 80% success rate when a frozen membrane precursor is brought to ambient temperature. This system will reduce laborious processes and expertise associated with ion channels.

An artificial lipid bilayer, or black lipid membrane (BLM), is a powerful tool for studying ion channels and protein interactions, as well as for ...

Single-Molecule Diffusion and Assembly on Polymer-Crowded Lipid Membranes

Cellular membranes are highly crowded environments for biomolecular reactions and signaling. Yet, most in vitro experiments probing protein interaction with lipids employ naked bilayer membranes. Such systems lack the complexities of crowding by membrane-embedded proteins and glycans and exclude the associated volume effects encountered on cellular membrane surfaces. Also, the negatively charged glass surface onto which the lipid bilayers are formed prevents the free diffusion of transmembrane biomolecules. Here, we present a well-characterized polymer-lipid membrane as a mimic for crowded lipid membranes. This protocol utilizes polyethylene glycol (PEG)-conjugated lipids as a generalized approach for incorporating crowders into the supported lipid bilayer&#160;(SLB). First, a cleaning procedure of the microscopic slides and coverslips for performing single-molecule experiments is presented. Next, methods for characterizing the PEG-SLBs and performing single-molecule experiments of the binding, diffusion, and assembly of biomolecules using single-molecule tracking and photobleaching are discussed. Finally, this protocol demonstrates how to monitor the nanopore assembly of bacterial pore-forming toxin Cytolysin A (ClyA) on crowded lipid membranes with single-molecule photobleaching analysis. MATLAB codes with example datasets are also included to perform some of the common analyses such as particle tracking, extracting diffusive behavior, and subunit counting.

Here, a protocol to perform and analyze the binding, mobility, and assembly of single molecules on artificial crowded lipid membranes using single-molecule total internal reflection fluorescence (smTIRF) microscopy is presented.

Cellular membranes are highly crowded environments for biomolecular reactions and signaling. Yet, most in vitro experiments probing protein interaction ...

Biochemistry

Temperature-Controlled Assembly and Characterization of a Droplet Interface Bilayer

The droplet interface bilayer (DIB) method for assembling lipid bilayers (i.e., DIBs) between lipid-coated aqueous droplets in oil offers key benefits versus other methods: DIBs are stable and often long-lasting, bilayer area can be reversibly tuned, leaflet asymmetry is readily controlled via droplet compositions, and tissue-like networks of bilayers can be obtained by adjoining many droplets. Forming DIBs requires spontaneous assembly of lipids into high density lipid monolayers at the surfaces of the droplets. While this occurs readily at room temperature for common synthetic lipids, a sufficient monolayer or stable bilayer fails to form at similar conditions for lipids with melting points above room temperature, including some cellular lipid extracts. This behavior has likely limited the compositions&#8212;and perhaps the biological relevance&#8212;of DIBs in model membrane studies. To address this problem, an experimental protocol is presented to carefully heat the oil reservoir hosting DIB droplets and characterize the effects of temperature on the lipid membrane. Specifically, this protocol shows how to use a thermally conductive aluminum fixture and resistive heating elements controlled by a feedback loop to prescribe elevated temperatures, which improves monolayer assembly and bilayer formation for a wider set of lipid types. Structural characteristics of the membrane, as well as the thermotropic phase transitions of the lipids comprising the bilayer, are quantified by measuring the changes in electrical capacitance of the DIB. Together, this procedure can aid in evaluating biophysical phenomena in model membranes over various temperatures, including determining an effective melting temperature (TM) for multi-component lipid mixtures. This capability will thus allow for closer replication of natural phase transitions in model membranes and encourage the formation and use of model membranes from a wider swath of membrane constituents, including those that better capture the heterogeneity of their cellular counterparts.

This protocol details the use of a feedback temperature-controlled heating system to promote lipid monolayer assembly and droplet interface bilayer formation for lipids with elevated melting temperatures, and capacitance measurements to characterize temperature-driven changes in the membrane.

The droplet interface bilayer (DIB) method for assembling lipid bilayers (i.e., DIBs) between lipid-coated aqueous droplets in oil offers key benefits ...

Assembly of Cell Mimicking Supported and Suspended Lipid Bilayer Models for the Study of Molecular Interactions

Model cell membranes are a useful screening tool with applications ranging from early drug discovery to toxicity studies. The cell membrane is a crucial protective barrier for all cell types, separating the internal cellular components from the extracellular environment. These membranes are composed largely of a lipid bilayer, which contains outer hydrophilic head groups and inner hydrophobic tail groups, along with various proteins and cholesterol. The composition and structure of the lipids themselves play a crucial role in regulating biological function, including interactions between cells and the cellular microenvironment, which may contain pharmaceuticals, biological toxins, and environmental toxicants. In this study, methods to formulate uni-lipid and multi-lipid supported and suspended cell mimicking lipid bilayers are described. Previously, uni-lipid phosphatidylcholine (PC) lipid bilayers as well as multi-lipid placental trophoblast-inspired lipid bilayers were developed for use in understanding molecular interactions. Here, methods for achieving both types of bilayer models will be presented. For cell mimicking multi-lipid bilayers, the desired lipid composition is first determined via lipid extraction from primary cells or cell lines followed by liquid chromatography-mass spectrometry (LC-MS). Using this composition, lipid vesicles are fabricated using a thin-film hydration and extrusion method and their hydrodynamic diameter and zeta potential are characterized. Supported and suspended lipid bilayers can then be formed using quartz crystal microbalance with dissipation monitoring (QCM-D) and on a porous membrane for use in a parallel artificial membrane permeability assay (PAMPA), respectively. The representative results highlight the reproducibility and versatility of in vitro cell membrane lipid bilayer models. The methods presented can aid in rapid, facile assessment of the interaction mechanisms, such as permeation, adsorption, and embedment, of various molecules and macromolecules with a cell membrane, helping in the screening of drug candidates and prediction of potential cellular toxicity.

This protocol describes the formation of cell mimicking uni-lipid and multi-lipid vesicles, supported lipid bilayers, and suspended lipid bilayers. These in vitro models can be adapted to incorporate a variety of lipid types and can be used to investigate various molecule and macromolecule interactions.

Model cell membranes are a useful screening tool with applications ranging from early drug discovery to toxicity studies. The cell membrane is a crucial ...

Multifunctional, Micropipette-based Method for Incorporation And Stimulation of Bacterial Mechanosensitive Ion Channels in Droplet Interface Bilayers

MscL, a large conductance mechanosensitive channel (MSC), is a ubiquitous osmolyte release valve that helps bacteria survive abrupt hypo-osmotic shocks. It has been discovered and rigorously studied using the patch-clamp technique for almost three decades. Its basic role of translating tension applied to the cell membrane into permeability response makes it a strong candidate to function as a mechanoelectrical transducer in artificial membrane-based biomolecular devices. Serving as building blocks to such devices, droplet interface bilayers (DIBs) can be used as a new platform for the incorporation and stimulation of MscL channels. Here, we describe a micropipette-based method to form DIBs and measure the activity of the incorporated MscL channels. This method consists of lipid-encased aqueous droplets anchored to the tips of two opposing (coaxially positioned) borosilicate glass micropipettes. When droplets are brought into contact, a lipid bilayer interface is formed. This technique offers control over the chemical composition and the size of each droplet, as well as the dimensions of the bilayer interface. Having one of the micropipettes attached to a harmonic piezoelectric actuator provides the ability to deliver a desired oscillatory stimulus. Through analysis of the shapes of the droplets during deformation, the tension created at the interface can be estimated. Using this technique, the first activity of MscL channels in a DIB system is reported. Besides MS channels, activities of other types of channels can be studied using this method, proving the multi-functionality of this platform. The method presented here enables the measurement of fundamental membrane properties, provides a greater control over the formation of symmetric and asymmetric membranes, and is an alternative way to stimulate and study mechanosensitive channels.

Bacterial mechanosensitive channels can be used as mechanoelectrical transducers in biomolecular devices. Droplet interface bilayers (DIBs), cell-inspired building blocks to such devices, represent new platforms to incorporate and stimulate mechanosensitive channels. Here, we demonstrate a new micropipette-based method of forming DIBs, allowing the study of mechanosensitive channels under mechanical stimulation.

MscL, a large conductance mechanosensitive channel (MSC), is a ubiquitous osmolyte release valve that helps bacteria survive abrupt hypo-osmotic shocks. ...

Planar Supported Lipid Bilayer Assay: A Microscopy-Based In Vitro Technique to Investigate Septin Assembly on Biological Membrane Mimics

Source: Curtis, B. N. et al. <a href="https://www-jove-com.bdigitaluss.remotexs.co/v/64090">Reconstitution of Septin Assembly at Membranes to Study Biophysical Properties and Functions.</a> J. Vis. Exp. (2022)
In this video, we demonstrate a procedure to investigate septin organization on a glass-supported lipid bilayer in vitro using total internal reflection fluorescence microscopy.

Source: Curtis, B. N. et al. Reconstitution of Septin Assembly at Membranes to Study Biophysical Properties and Functions. J. Vis. Exp. (2022)
In this ...