Name: Single-Channel Electrophysiology Using a Miniaturized Montal–Mueller Planar Lipid Bilayer System for Recording a Synthetic K2P Channel (Video) | JoVE Journal
Uploaded: 2026-10-09T13:05:00.000+00:00
Duration: 7 min 58 s
Description: Learn single-channel electrophysiology using a miniaturized Montal–Mueller planar lipid bilayer system for recording ion channels. This method enables stable analysis of synthetic K2P channels with reduced reagent consumption.

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We thank Sofia Diletta Guzzeloni for helping with the cell transfection procedure. We are grateful to Elements employees Dr. Leon Juarez and Dr. Michelangelo Paci. This work was supported by the Department of Molecular Medicine of the University of Pavia, Italy under the initiative Dipartimenti di Eccellenza (2023&#8211;2027), by the Cariplo Foundation (#2019-1809) and by PNRR Research Programme CN00000013 &#8220;National Centre for HPC, Big Data and Quantum Computing&#8221; to M.L.

F.T. and A.P. are the CEO and an employee of Elements S.r.l., respectively. All other authors declare no competing interests.

In this study, we used the PLB technique to evaluate the electrophysiological properties of a synthetic K2P channel at the single-molecule level. Among the various planar lipid models described in the scientific literature, we focused on the formation of BLMs, which are painted over an aperture with a diameter between 50 and 140 &#181;m, drilled on a vertical non-polar surface separating two aqueous compartments. This configuration allows simulation of the cell membrane environment while maintaining control over experimental parameters, including lipid membrane preparation and electrolyte composition (e.g., ionic strength and pH)3. We recreated BLMs using the Montal&#8211;Mueller (folding) method5, in which the membrane assembles spontaneously when two lipid monolayers, previously spread at the air&#8211;water interface of the two aqueous solutions, join over the aperture by raising the water level.
The protocol presented here for membrane formation is highly intuitive; however, several critical steps strongly influence experimental success. To ensure proper electrical connection between the BLMchipMM and the amplifier, it is essential to avoid chlorination of the Ag/AgCl wire ends that are inserted into the PCB sockets. During BLMchipMM assembly, improper positioning of the wires may result in contact with the amplifier case, leading to short circuits and signal saturation. The aperture pre-treatment procedure is another critical factor: excessive force during application of the pre-treatment solution may damage the polyimide septum, while improper handling of the electrolyte solution&#8212;particularly soaking the aperture region&#8212;can impair membrane formation. Furthermore, careful cleaning of the BLMchipMM recording chamber is essential for reproducibility. The use of 100% isopropyl alcohol followed by thorough rinsing and complete drying ensures that residues do not interfere with subsequent experiments.
Several modifications to the protocol are possible. While chlorination by bleach soaking represents a practical and widely used approach, alternative methods such as electroplating can also be employed43. Similarly, variations in pre-treatment and lipid preparation are feasible. Standard pre-treatment solutions typically consist of long-chain alkanes dissolved in volatile hydrocarbon solvents36; although a 1% hexadecane solution in n-hexane is commonly used, other solvents may be suitable. Lipid composition can also be adapted depending on the experimental requirements. While DPhPC lipids provide optimal membrane stability, alternative lipid mixtures, including natural or synthetic compositions, can be used36. A key advantage of vertical lipid bilayers formed via the folding method is the ability to generate asymmetric membranes, where each leaflet consists of different lipid species5,22, a feature known to influence membrane protein function.
Troubleshooting membrane formation generally involves adjustments of the aqueous phase levels. If membrane formation does not occur initially, repeated lowering and raising of the buffer level in one compartment can facilitate bilayer assembly. If unsuccessful, applying an air bubble over the aperture can promote lipid redistribution and membrane formation. Persistent failure typically indicates issues related to pre-treatment or contamination, in which case cleaning the recording chamber and restarting the experiment is recommended.
The core components of this setup comprise the eNPR 100 kHz compact amplifier and BLMchipMM. The amplifier is a compact, low-noise voltage-clamp system that eliminates the need for additional external devices, including external Faraday cages, as the amplifier casing itself provides effective electromagnetic shielding. The BLMchipMM is a pre-assembled, miniaturized recording chamber integrated with a PCB for direct connection to the amplifier. It consists of two C-shaped compartments separated by a polyimide septum with a tapered laser-drilled aperture where the artificial membrane is formed. The reduced dimensions of the system, with compartment volumes of approximately 110 &#181;L, significantly decrease reagent consumption and reduce intrinsic capacitance, thereby improving signal quality. In addition, the device can be reused multiple times without disassembly when proper cleaning procedures are followed, reducing variability and enhancing experimental reproducibility.
Despite these advantages, some limitations should be considered. The current setup does not include integrated modules for temperature, pressure, or perfusion control. Intrinsic membrane tension has been reported to influence mechanosensitive channels such as TREK144,45,46; however, the present setup does not include controlled manipulation of membrane tension, and no mechanistic conclusions are drawn here. Overall, this platform provides a robust and accessible system for recording ion channel activity in artificial membranes. In this study, we investigated the synthetic K2P channel ancTREK1, a computationally derived ancestral channel of the TREK subfamily. The recorded data demonstrate that purified ancTREK1 reconstituted into PLBs exhibits single-channel behavior consistent with reported TREK1-like conductance properties. Under Mg2+-free symmetric 200 mM potassium conditions, the channel displays the measurable I&#8211;V relationship, with amplitude values of approximately 13 pA at 80 mV and &#8722;19 pA at &#8722;80 mV. Additionally, ancTREK1 exhibits the characteristic large conductance mode observed in TREK1 channels, with higher conductance at negative potentials (~205.6 pS) compared to positive potentials (~112.8 pS). Notably, ancTREK1 shows an increased NPo at positive potentials (0.78 at 80 mV) and reduced NPo at negative potentials (0.53 at &#8722;80 mV and 0.28 at &#8722;120 mV), differing from observations in excised patch configurations42. These findings support the use of this system for proof-of-principle single-channel recording of reconstituted membrane proteins.

The reconstitution of ion channels into artificial lipid bilayer membranes, also known as black lipid membranes (BLMs), is one of the established techniques to perform electrophysiological characterization of ion channels at the single-molecule level1. The advantages of BLMs arise from their structure. Indeed, a planar lipid bilayer (PLB) is created within a pore in a non-polar surface, separating two liquid solutions. Thus, by mimicking a biological membrane, it allows the recording of ion channel activity under highly controlled conditions, including membrane lipid composition and electrolyte or chemical compound concentrations, while remaining accessible from both sides. This accessibility also allows ion channels to be directly inserted, for example from a micellar solution or by liposome-driven fusion2,3.
The first artificial membrane system to be described was created using the painting technique4, where a droplet of lipids dissolved in a non-volatile organic solvent is applied to a small aperture with a paintbrush. The addition of an aqueous solution to both sides of the aperture then leads to the spontaneous formation of a lipid bilayer. However, a significant disadvantage of this method is the retention of solvent traces, which may affect the membrane&#8217;s properties. To reduce the amount of residual organic solvent within lipid bilayers, the folding method5 was developed, enabling the formation of &#8220;solvent-free&#8221; BLMs. In this approach, a lipid solution in a volatile solvent is spread on the water surface to form two monolayers, and by gradually raising the level of the aqueous phase above the aperture, these lipid monolayers fold over the aperture to form a bilayer. However, although the resulting BLM is defined as solvent-free, successful bilayer formation requires pre-treating the aperture with a thin layer of non-volatile solvent. The core element required for these experiments consists of a recording chamber with two aqueous compartments (typically referred to as cis and trans) separated by a lipophilic septum with a small aperture (50&#8211;250 &#181;m in diameter) (Figure 1). Silver/silver chloride (Ag/AgCl) electrodes are inserted into each compartment. When immersed in the electrolyte solution, these electrodes establish the electrochemical connection between the lipid bilayer and the recording apparatus. The latter consists of a voltage-clamp amplifier, which enables precise control of the transmembrane potential and simultaneous measurement of the ionic currents passing through embedded pore-forming proteins.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/69903/69903fig1.jpg" /> 
Figure 1: Schematic of planar lipid bilayer (PLB) electrophysiology setup. A schematic of the essential components required for PLB experiments. The recording chamber consists of two aqueous compartments (cis and trans) separated by a lipophilic septum containing an aperture where a lipid bilayer is formed. Silver/silver chloride (Ag/AgCl) electrodes are immersed in the electrolyte solution in each compartment. One electrode applies voltage stimuli (e.g., sine, square, or triangular waveforms), while the other, held at ground, connects to a current-to-voltage converter that transforms ionic current into a proportional voltage signal. The signal is amplified, optionally filtered, digitized, and recorded for real-time monitoring and analysis. <a href="https://www.jove.com/files/ftp_upload/69903/69903fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Other methods have also been established to form BLMs. Among these, a common approach consists of creating lipid bilayers suspended on the tip of a patch-clamp pipette. Accordingly, the tip-dip method6 allows the formation of a suspended solvent-free lipid bilayer on the aperture of a glass micropipette with a diameter of ~4 &#181;m by repeatedly dipping the tip through a lipid monolayer at the air&#8211;water interface, thereby capturing a single leaflet during each passage. The patch-clamp technique has also been used for single-channel measurements in lipid bilayers. For example, in the punch-out method7, a glass pipette with a diameter between 10 and 80 &#181;m is used to patch a large membrane originally painted over an aperture in a recording chamber. Alternatively, the liposome-patch method involves the use of a pre-formed nano- to microsized vesicle as a solvent-free BLM8,9. In particular, giant unilamellar vesicles have been widely used for single-channel recordings through different patch-clamp configurations, including cell-attached and excised patch modes10,11.
Several platforms have also emerged for the parallel and automated formation of multiple bilayers. One such approach is the droplet interface bilayer (DIB) technique12, in which aqueous droplets are placed in a lipid&#8211;solvent mixture. In this method, lipid monolayers spontaneously assemble around the droplets, and bilayer formation occurs when two droplets come into contact. In some setups, DIBs can be formed on hydrogels to improve membrane stability13. An alternative approach based on DIB formation at the tip of a glass pipette is the contact bubble bilayer (CBB) method14, where aqueous bubbles are introduced into an oil phase using two micropipettes. As in the DIB approach, a lipid bilayer spontaneously forms when two lipid-coated bubbles are brought into contact. Another platform is represented by arrayed BLM systems, which consist of arrays of well-defined micro- to nanoscale pores arranged in a grid pattern. In these systems, suspended lipid bilayers can be formed either manually&#8212;using painting or folding techniques, or via liposome vesicle rupture15,16,17&#8212;or automatically by sequential injection of lipid and buffer solutions when coupled with microfluidic channel structures18,19.
Although many methods have been developed to form solvent-free BLMs, in this work we focus on the folding method implemented within a miniaturized and integrated platform to investigate single-channel behavior of membrane proteins embedded in an artificial lipid bilayer. Experiments were performed using the Elements setup, which consists of two components: the Elements Nanopore Reader (eNPR amplifier) and the BLMchipMM (Figure 2A and Figure 2B). The former is a compact, low-noise voltage-clamp amplifier that simplifies the workspace by forming a complete and independent unit, eliminating the need for additional external devices. The BLMchipMM is a pre-assembled and miniaturized system designed for the Montal&#8211;Mueller technique and serves as the experimental interface. It consists of a transparent polymethyl methacrylate (PMMA) recording chamber integrated with a printed circuit board (PCB), which includes two sockets designed to accommodate Ag/AgCl wires serving as electrodes for direct connection to the amplifier. The core structure of the chamber consists of a polyimide septum sealed between two transparent PMMA components. The septum is 50 &#181;m thick, except in a small central region (3.78 mm2), where the thickness is reduced to 25 &#181;m to facilitate bilayer formation. At the center of this thinner region, a tapered aperture is created by laser drilling, which enhances the mechanical stability of the lipid bilayer20,21. Four aperture diameters (50, 75, 100, and 140 &#181;m) are available, enabling tuning of membrane capacitance (Table 1). Each PMMA component features a C-shaped cavity with a volume of 110 &#181;L, forming two fluidic compartments on either side of the polyimide septum to hold the electrolyte solution. Two tilted fluidic channels are drilled into each PMMA block on opposite sides: one channel is used to insert the Ag/AgCl electrode, while the other allows manual adjustment of the buffer level within the reservoir by pipetting.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/69903/69903fig2.jpg" /> 
Figure 2: Integrated Elements Nanopore Reader and BLMchipMM system. (A) Photograph showing the BLMchipMM inserted into the dedicated slot of the Elements Nanopore Reader (eNPR) amplifier. (B) Schematic representation of the BLMchipMM components, including the polymethyl methacrylate (PMMA) recording chamber, the polyimide septum containing the micro-sized aperture, and the printed circuit board (PCB) with sockets for Ag/AgCl electrodes and a slot for chamber alignment. <a href="https://www.jove.com/files/ftp_upload/69903/69903fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Hole diameter (&#181;m)</td>
			<td>50</td>
			<td>75</td>
			<td>100</td>
			<td>140</td>
		</tr>
		<tr>
			<td>Hole thickness (&#181;m)</td>
			<td>25</td>
			<td>25</td>
			<td>25</td>
			<td>25</td>
		</tr>
		<tr>
			<td>Typical membrane capacitance (pF)</td>
			<td>20&#8211;40</td>
			<td>40&#8211;60</td>
			<td>60&#8211;90</td>
			<td>90&#8211;140</td>
		</tr>
	</tbody>
</table>
Table 1: Characteristics of available BLMchipMM aperture formats. Hole diameter, septum thickness at the aperture region, and typical membrane capacitance values for BLMchipMM devices with 50, 75, 100, and 140 &#181;m apertures. Membrane capacitance values are shown for bilayers formed from 1,2-diphytanoyl-sn-glycero-3-phosphocholine lipids.
Typically, recording chambers used in laboratories are either homemade units or commercially available devices. In the former case, constructing the system involves assembling various components and developing a reliable method to create the aperture in the septum, which can limit accessibility for many laboratories. Even when commercial devices are used, they often require manual integration of multiple components prior to each experiment. In contrast, the BLMchipMM is pre-assembled and does not require disassembly between experiments for routine cleaning procedures. The same unit can be washed and reused for multiple experiments without compromising membrane formation quality, provided that the solvents used are compatible with the device materials. This reduces user-dependent variability and minimizes the risk of leakage associated with manual sealing elements. The use of external, replaceable, and reconditionable silver wire electrodes further extends the usable lifetime of the system, especially under experimental conditions that accelerate electrode consumption.
Moreover, homemade devices often feature large compartment volumes, increasing the amount of biological and chemical components required for experiments. In contrast, the miniaturization of the BLMchipMM allows the minimization of experimental volumes, thereby significantly reducing the consumption of reagents, proteins, and other compounds. This is particularly advantageous when working with difficult-to-express or low-yield membrane proteins, or when the high cost of synthesizing test compounds increases the overall cost of experiments. Importantly, the BLMchipMM enables the formation of solvent-minimized bilayers and supports asymmetric membrane configurations, where each leaflet can be composed of different lipid species5,22, facilitating the study of membrane protein function under controlled conditions.
Traditionally, current measurements are performed using amplifiers designed for patch-clamp experiments, which often require connection to a separate digitizer for data acquisition and control via graphical interfaces. These systems are not only bulky and expensive, but also frequently involve non-intuitive setup procedures for non-expert users. Additionally, due to the size of the recording chamber and the design of standard amplifiers&#8212;which typically lack built-in shielding to protect the headstage input from electrostatic interference&#8212;the use of an external Faraday cage is often necessary. The quality and grounding of such a cage can significantly affect the system&#8217;s noise performance. The BLMchipMM, when used with a compact integrated amplifier, simplifies this configuration by reducing the need for external components. Signal digitization occurs within the device, and the system housing provides shielding that reduces susceptibility to ambient electromagnetic interference.
To demonstrate the applicability of this platform, the K2P-like channel ancTREK1, a computationally derived ancestral member of the TREK subfamily (Supplementary Figure 1), was used as a test protein to validate successful reconstitution and single-channel recording. K2P channels form dimeric potassium leak channels that regulate resting membrane potential and cellular excitability, with TREK channels representing a well-characterized subfamily.

<table><tbody><tr><td>1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC)</td><td>Avanti Polar Lipids</td><td>850356P</td><td>Formation of artificial lipid bilayers</td></tr><tr><td>2-Propanol (&amp;ge;99.5%)</td><td>Honeywell</td><td>24137-5L</td><td>Cleaning of BLMchipMM device</td></tr><tr><td>Agarose</td><td>Fisher BioReagents</td><td>BP160-500</td><td>Preparation of agarose gels</td></tr><tr><td>Agarose gel horizontal electrophoresis system</td><td>BTLabSystems</td><td>BT102</td><td>Separation of DNA fragments</td></tr><tr><td>Ampicillin</td><td>Fisher BioReagents</td><td>BP1760-25</td><td>Selection of transformed bacteria</td></tr><tr><td>Anti-GFP nanobody Sepharose resin (homemade)</td><td>&amp;mdash;</td><td>&amp;mdash;</td><td>Affinity purification of GFP-tagged proteins</td></tr><tr><td>Anza Alkaline Phosphatase</td><td>Invitrogen</td><td>IVGN2208</td><td>Vector dephosphorylation</td></tr><tr><td>Anza EcoRI</td><td>Invitrogen</td><td>IVGN0116</td><td>Restriction digestion</td></tr><tr><td>Anza NotI</td><td>Invitrogen</td><td>IVGN0014</td><td>Restriction digestion</td></tr><tr><td>Anza Red Buffer (10&amp;times;)</td><td>Invitrogen</td><td>IVGN2008</td><td>Restriction digestion buffer</td></tr><tr><td>Aprotinin protease inhibitor</td><td>Fisher Scientific</td><td>15996008</td><td>Prevention of protein degradation</td></tr><tr><td>Benchtop centrifuge</td><td>Eppendorf</td><td>EP5420000164</td><td>Cell pelleting and sample preparation</td></tr><tr><td>BLMchipMM</td><td>Elements s.r.l.</td><td>&amp;mdash;</td><td>Formation of planar lipid bilayers</td></tr><tr><td>Cholesteryl hemisuccinate (CHS)</td><td>Anatrace</td><td>CH210</td><td>Stabilization of membrane proteins</td></tr><tr><td>Coomassie Brilliant Blue</td><td>Sigma-Aldrich</td><td>1125530025</td><td>Protein staining</td></tr><tr><td>Common household bleach</td><td>&amp;mdash;</td><td>&amp;mdash;</td><td>Chlorination of Ag wires</td></tr><tr><td>DNase I</td><td>Sigma-Aldrich</td><td>11284932001</td><td>Reduction of lysate viscosity</td></tr><tr><td>Double distilled water</td><td>&amp;mdash;</td><td>&amp;mdash;</td><td>Preparation of buffers and solutions</td></tr><tr><td>Ethanol (pure, 99.9%)</td><td>Girelli Alcool s.r.l.</td><td>&amp;mdash;</td><td>Cleaning of Ag/AgCl wires</td></tr><tr><td>Ethidium bromide</td><td>Sigma-Aldrich</td><td>1116080030</td><td>DNA staining</td></tr><tr><td>Extra fine grit sandpaper</td><td>&amp;mdash;</td><td>&amp;mdash;</td><td>Removal of Ag/AgCl coating from wires</td></tr><tr><td>eNPR 100 kHz amplifier</td><td>Elements s.r.l.</td><td>&amp;mdash;</td><td>Electrophysiological recordings</td></tr><tr><td>Gel-Out kit for DNA extraction from agarose</td><td>A&amp;amp;A Biotechnology</td><td>023-250</td><td>DNA purification</td></tr><tr><td>GeneRuler 1 kb DNA ladder</td><td>Thermo Fisher Scientific</td><td>SM0311</td><td>DNA size reference</td></tr><tr><td>FreeStyle&amp;trade; 293-F</td><td>Thermo Fisher Scientific</td><td>R79007</td><td>Expression of ancTREK1 protein</td></tr><tr><td>HEPES (&amp;ge;99.5%)</td><td>Sigma-Aldrich</td><td>H3375-500G</td><td>Buffering agent</td></tr><tr><td>Hexadecane (&amp;ge;99%)</td><td>Sigma-Aldrich</td><td>H6703-100ML</td><td>Aperture pre-treatment</td></tr><tr><td>Hexane (&amp;ge;99%)</td><td>Sigma-Aldrich</td><td>139386-100ML</td><td>Lipid solvent</td></tr><tr><td>Homemade 5&amp;times; DNA loading dye</td><td>&amp;mdash;</td><td>&amp;mdash;</td><td>DNA sample preparation</td></tr><tr><td>Homemade 6&amp;times; Laemmli SDS buffer (reducing)</td><td>&amp;mdash;</td><td>&amp;mdash;</td><td>Protein sample preparation</td></tr><tr><td>Homemade destain solution</td><td>&amp;mdash;</td><td>&amp;mdash;</td><td>Destaining SDS-PAGE gels</td></tr><tr><td>Homemade HRV-3C protease</td><td>&amp;mdash;</td><td>&amp;mdash;</td><td>Cleavage of fusion proteins</td></tr><tr><td>Homemade SDS-PAGE running buffer (10&amp;times;)</td><td>&amp;mdash;</td><td>&amp;mdash;</td><td>SDS-PAGE electrophoresis buffer</td></tr><tr><td>Imidazole</td><td>Alfa Aesar</td><td>A10221</td><td>Elution of His-tagged proteins</td></tr><tr><td>In-batch purification columns</td><td>&amp;mdash;</td><td>&amp;mdash;</td><td>Protein purification workflows</td></tr><tr><td>Innova44 shaking incubator</td><td>New Brunswick Innova</td><td>M1282</td><td>Cell culture growth</td></tr><tr><td>KCl</td><td>Honeywell</td><td>31248-1KG</td><td>Electrolyte and buffer preparation</td></tr><tr><td>LB broth (Lennox)</td><td>Condalab</td><td>1231</td><td>Bacterial culture</td></tr><tr><td>Leupeptin protease inhibitor</td><td>Thermo Scientific</td><td>78435</td><td>Prevention of protein degradation</td></tr><tr><td>Magnesium chloride hexahydrate</td><td>Acros Organics</td><td>447700010</td><td>Buffer component</td></tr><tr><td>Nanodrop spectrophotometer</td><td>DeNovix</td><td>DS-11</td><td>DNA and protein quantification</td></tr><tr><td>n-Dodecyl-&amp;beta;-D-maltoside (DDM)</td><td>NeoBiotech</td><td>NB-19-0056-25G</td><td>Membrane protein solubilization</td></tr><tr><td>Ni Sepharose High Performance Resin</td><td>Cytiva</td><td>17526801</td><td>IMAC purification</td></tr><tr><td>Notebook (computer)</td><td>&amp;mdash;</td><td>&amp;mdash;</td><td>Instrument control and data acquisition</td></tr><tr><td>Optima LE-80K ultracentrifuge</td><td>Beckman</td><td>365668</td><td>Membrane fraction isolation</td></tr><tr><td>Pentane (&amp;ge;99%)</td><td>Sigma-Aldrich</td><td>236705-100ML</td><td>Lipid solvent</td></tr><tr><td>Pepstatin A protease inhibitor</td><td>Thermo Scientific</td><td>78436</td><td>Prevention of protein degradation</td></tr><tr><td>Phusion GC Buffer (5&amp;times;)</td><td>Thermo Fisher Scientific</td><td>F519</td><td>PCR buffer</td></tr><tr><td>Phusion High-Fidelity DNA Polymerase</td><td>Thermo Fisher Scientific</td><td>F530S</td><td>PCR amplification</td></tr><tr><td>PMSF protease inhibitor</td><td>Thermo Scientific</td><td>36978</td><td>Protein protection during lysis</td></tr><tr><td>Polyethylenimine (PEI)</td><td>Clinisciences</td><td>24765-100</td><td>Transfection reagent</td></tr><tr><td>ProteinEle precast Tris-Glycine gel (4&amp;ndash;20%)</td><td>TransGen Biotech</td><td>DG101-01-V2</td><td>Protein separation</td></tr><tr><td>SDS-PAGE gel electrophoresis system</td><td>Bio-Rad</td><td>1658002FC</td><td>Protein electrophoresis</td></tr><tr><td>Sodium butyrate</td><td>Fisher Scientific</td><td>A11079</td><td>Enhancer of protein expression</td></tr><tr><td>T4 DNA ligase</td><td>Thermo Fisher Scientific</td><td>EL0011</td><td>DNA ligation</td></tr><tr><td>T4 DNA ligase buffer (10&amp;times;)</td><td>Thermo Fisher Scientific</td><td>B69</td><td>Ligation buffer</td></tr><tr><td>Thermal cycler (Mastercycler Nexus)</td><td>Eppendorf</td><td>6331000025</td><td>PCR amplification</td></tr><tr><td>Tris base</td><td>Sigma-Aldrich</td><td>T1503-1KG</td><td>Buffer preparation</td></tr><tr><td>Tryptone SOYA agar with lecithin and polysorbate 80</td><td>HiMedia</td><td>M449-5KG</td><td>Bacterial plating</td></tr><tr><td>Two Ag wires</td><td>Elements s.r.l.</td><td>&amp;mdash;</td><td>Electrodes for electrophysiology</td></tr><tr><td>Ultra-spin concentrator (50 kDa cutoff)</td><td>Merck Amicon</td><td>UFC8050</td><td>Protein concentration</td></tr><tr><td>pEG BacMam expression vector</td><td>Addgene</td><td>160687</td><td>Protein expression</td></tr><tr><td>USB cable</td><td>&amp;mdash;</td><td>&amp;mdash;</td><td>Connection between amplifier and computer</td></tr></tbody></table>

single-channel electrophysiology using a miniaturized montal&#8211;mueller planar lipid bilayer system for recording a synthetic k2p channel

Single-Channel Electrophysiology Using a Miniaturized Montal&#8211;Mueller Planar Lipid Bilayer System for Recording a Synthetic K2P Channel

The functional reconstitution of ion channels into planar lipid bilayers provides a strategy to investigate channel activity at the single-molecule level. However, conventional Montal&#8211;Mueller bilayer systems typically require bulky experimental setups, manual chamber assembly, and relatively large recording volumes, which increase reagent consumption and may introduce additional sources of electrical noise due to extended wiring and external headstages. Here, we present a miniaturized, ready-to-use bilayer platform (BLMchipMM) integrated with a compact amplifier that enables stable single-channel recordings with reduced reagent consumption and simplified operation. This protocol describes the use of this system for performing single-channel electrophysiology measurements. Using this system, we reconstituted a synthetic K2P channel (ancTREK1) and used it as a proof-of-principle test protein to validate reconstitution and single-channel recording. Single-channel recordings show a current&#8211;voltage relationship in Mg2+-free symmetric K+ solutions, with conductance values consistent with those reported for TREK1 channels. The successful reconstitution of this K2P-like channel demonstrates the utility of this system for single-channel analysis in artificial membranes.

Current&#8211;voltage (I&#8211;V) characteristics of PLB membranes
The intrinsic hydrophobic nature of bilayer membranes makes them impermeable to charged species. However, the application of an external electric field can induce the formation of transient pores of nanometer size, thereby affecting membrane permeability and conductance33. To assess the quality of artificial bilayers formed from 1,2-diphytanoyl-sn-glycero-3-phosphocholine lipids, membrane capacitance, resistance, and I&#8211;V characteristics were evaluated in the absence of inserted proteins. A series of voltage steps ranging from &#177;20 to &#177;200 mV (in &#177;20 mV increments) was applied, and the resulting transmembrane currents were recorded. Data were sampled at 5 kHz and low-pass filtered at 2.5 kHz.
Membrane capacitance (Cm) and membrane resistance (Rm) represent key parameters for evaluating bilayer quality. The PLB can be described by an equivalent resistor&#8211;capacitor (RC) circuit, in which the membrane behaves as a capacitor in parallel with a resistor34. The capacitive component reflects the membrane&#8217;s ability to store charge, whereas the resistive component reflects ion permeability. The application of a triangular voltage waveform generates a square-shaped capacitive current response, which is proportional to the membrane capacitance according to:
<img alt="Equation 1" src="/files/ftp_upload/69903/69903eq1.jpg" style="margin: auto;" />&#160; &#160; (1)
Here, Cm is the membrane capacitance, V is the applied voltage, and t is time. Following membrane formation, capacitance values can increase over time due to progressive thinning of the bilayer35. Considering the membrane as a parallel plate capacitor, capacitance is related to membrane area and thickness according to:
<img alt="Equation 2" src="/files/ftp_upload/69903/69903eq2.jpg" style="margin: auto;" />&#160; &#160; (2)
Here, &#949;lipids is the dielectric constant of the lipid core (~2), &#949;0 is the permittivity of free space, A is membrane area, and d is membrane thickness36,37.
Membrane resistance was determined using Ohm&#8217;s law33:
<img alt="Equation 3" src="/files/ftp_upload/69903/69903eq3.jpg" style="margin: auto;" />&#160; &#160; (3)
Here, V is the applied voltage and Rm is the membrane resistance. In the absence of ion channels, the membrane current remains near 0 pA, corresponding to resistance values in the giga-ohm (G&#937;) range33.
The I&#8211;V relationship exhibited linear (ohmic) behavior between 0 and &#177;200 mV for 75 and 100 &#181;m aperture BLMchipMM devices. Linear fitting of the data yielded membrane resistance values of 847 &#177; 16 G&#937; (n = 6) for the 75 &#181;m aperture (Figure 8A, circles) and 980 &#177; 32 G&#937; (n = 7) for the 100 &#181;m aperture (Figure 8A, squares). RMS noise values, measured over 3 s segments of the current trace, were 3.1 &#177; 2 pA (n = 7) for the 100 &#181;m aperture and 2.6 &#177; 0.1 pA (n = 6) for the 75 &#181;m aperture. Membrane stability was assessed by measuring resistance at approximately 2 min intervals over a total duration of 1 h (Figure 8B). Well-formed membranes consistently remained stable for over 1 h, which is sufficient for most electrophysiological experiments. Membranes typically tolerated applied potentials up to &#177;200 mV without signs of instability, whereas breakdown occurred between &#177;220 and &#177;300 mV depending on the individual bilayer. Membranes were considered suitable for protein insertion when capacitance values ranged from 40 to 60 pF for 75 &#181;m apertures and from 60 to 90 pF for 100 &#181;m apertures, with current values of &#8804;0.5 pA at 100 mV.
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/69903/69903fig8.jpg" /> 
Figure 8: Electrical characterization of PLBs. (A) Current&#8211;voltage (I&#8211;V) relationships of bilayers formed on 75 &#181;m and 100 &#181;m apertures, demonstrating linear (ohmic) behavior between &#177;200 mV. Linear regression yields membrane resistance values of 847 &#177; 16 G&#937; (n = 6) and 980 &#177; 32 G&#937; (n = 7), respectively. (B) Membrane stability assessed by monitoring resistance over 1 h at 2 min intervals. Membranes remain stable up to &#177;200 mV and rupture at voltages between &#177;220 and &#177;300 mV. Data are presented as mean &#177; standard error of the mean (SEM). <a href="https://www.jove.com/files/ftp_upload/69903/69903fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Initial characterization of ancTREK1 single-channel behavior
Synthetic ancTREK1 was purified and reconstituted into PLBs, and single-channel activity was monitored using voltage step protocols ranging from &#8722;120 to 120 mV in 20 mV increments. Single-channel recordings (Figure 9A and Figure 9B) revealed the characteristic flickering behavior of K2P channels38. At 80 mV, the mean current amplitude was 13.02 &#177; 0.78 pA (n = 5), whereas at &#8722;80 mV the current amplitude increased to &#8722;18.72 &#177; 0.25 pA (n = 5) (Figure 9C). The resulting I&#8211;V relationship showed measurable single-channel currents at both positive and negative potentials under Mg2+-free symmetric 200 mM potassium conditions (Figure 9D). Because current amplitudes were estimated from all-point histograms of recordings acquired at 5 kHz and low-pass filtered at 2.5 kHz, this I&#8211;V relationship is presented as a descriptive proof-of-principle rather than as an exhaustive biophysical characterization.
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/69903/69903fig9v2.jpg" /> 
Figure 9: Single-channel activity of synthetic ancTREK1. (A and B) Representative single-channel current traces recorded at 80 mV and &#8722;80 mV, respectively. Dotted lines indicate the closed-channel level. (C) All-point amplitude histograms for positive (left) and negative (right) potentials. (D) I&#8211;V relationship of ancTREK1 under symmetric 200 mM potassium (K+) conditions without divalent cations. The curve is fitted using a fourth-order polynomial, and conductance values are derived from the slope at &#177;80 mV. <a href="https://www.jove.com/files/ftp_upload/69903/69903fig9largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Although ancTREK1 lacks extended N- and C-terminal regions compared to mammalian TREK1 channels, it remained functionally active upon reconstitution. In contrast to mammalian TREK1, which exhibits high- and low-conductance modes (132 &#177; 5 pS and 41 &#177; 5 pS at positive potentials, respectively)39, ancTREK1 exhibited only the large conductance mode. The single-channel conductance at positive potentials was 112.8 &#177; 10.8 pS (80 mV, n = 5), consistent with reported values for TREK channels (85&#8211;130 pS under symmetric potassium conditions)38,39,40,41. At negative potentials, conductance increased to 205.6 &#177; 2.05 pS (&#8722;80 mV, n = 5), in agreement with previously reported TREK1 behavior39. Notably, ancTREK1 exhibited a higher normalized open probability (NPo) compared to TREK1 measurements obtained using excised patch configurations42. The NPo&#8211;voltage relationship showed increased channel activity at positive potentials, reaching 0.78 &#177; 0.02 (n = 5) at 80 mV. In contrast, NPo decreased at negative potentials, with values of 0.53 &#177; 0.04 at &#8722;80 mV and 0.28 &#177; 0.03 at &#8722;120 mV (Figure 10).
<img alt="Figure 10" class="xfigimg" src="/files/ftp_upload/69903/69903fig10.jpg" /> 
Figure 10: Voltage dependence analysis of ancTREK1 single-channel activity. Normalized open probability (NPo) as a function of membrane potential (left) and comparison of NPo at 80 mV and &#8722;80 mV (right), showing increased channel opening at positive potentials. <a href="https://www.jove.com/files/ftp_upload/69903/69903fig10large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Figure 1. Amino acid sequence of the synthetic ancestral two-pore domain potassium (K2P) channel (ancTREK1). The full-length amino acid sequence of ancTREK1 used for electrophysiological recordings. The sequence was reconstructed by ancestral sequence inference from a curated dataset of TREK/TRAAK family members, including orthologous sequences collected from UniProt and NCBI databases, filtered for conserved pore motifs, aligned, and analyzed using maximum-likelihood phylogenetics.<a href="https://www.jove.com/files/ftp_upload/69903/Supplementary Figure 1.pdf" target="_blank">Please click here to download this file.</a>

This protocol describes the use of a miniaturized Montal&#8211;Mueller planar lipid bilayer system with integrated amplification to perform stable single-channel recordings. The method is demonstrated by reconstituting a purified synthetic K2P channel to validate system performance.

The functional reconstitution of ion channels into planar lipid bilayers provides a strategy to investigate channel activity at the single-molecule level. ...

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Biochemistry

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Learn single-channel electrophysiology using a miniaturized Montal–Mueller planar lipid bilayer system for recording ion channels. This method enables stable analysis of synthetic K2P channels with reduced reagent consumption.

Single-Channel Electrophysiology Using a Miniaturized Montal–Mueller Planar Lipid Bilayer System for Recording a Synthetic K2P Channel (Video) | JoVE Journal

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