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Biochemistry & Molecular Biology
The bilayer lipid membrane (BLM) is the main structural component of cell membranes, in which various membrane proteins are embedded. Artificially formed BLMs have been used as a platform in studies of the functions of membrane proteins, including various ion channels.
- bilayer lipid membranes
- ion channel
- lateral voltage
1. Introduction
The cell is surrounded by a membrane composed of bilayer lipid membranes (BLMs) and membrane proteins. The BLM has an ultrahigh resistance, higher than 1 TΩ [1], serving as a barrier for the permeation of ions across the cell membrane. Among the various membrane proteins, ion channels regulate transmembrane ion flow and play a key role in various physiological processes, including in the generation of the action potential, nerve transmission, and heartbeat [2]. Owing to their physiological importance, ion channels are also major targets for drug design [3]. In contrast to recent drastic progress in the structural analysis of ion channels due to the advent of the cryo-electron microscope [4,5], progress in the functional analysis of ion channels has remained moderate, with the patch-clamp method (awarded the Nobel Prize in 1991) still being the gold standard. There is a great demand for a novel functional analysis system that could provide new insights into ion channels in terms of physiological and pharmaceutical aspects.
The reconstitution of ion channel proteins in artificially formed BLMs is another approach for recording ion channel activities [6]. BLMs with channels imbedded in them can be regarded as cell-membrane-mimicking systems that allow the buffer and membrane conditions to be controlled at a higher level than actual cell membranes. Combined with the rapidly evolving synthetic biology using cell-free protein expression technologies [7,8], researchers can, in principle, design BLMs that contain only one channel genotype, leading to the development of a next-generation drug-screening platform. In addition, BLM systems also allow innovative experimental settings that are impossible with cell membranes. For example, when we place additional electrodes in the BLM interior in addition to the two conventional electrodes for transmembrane voltages, the additional electrodes can introduce a new input, namely a lateral voltage, to the BLM system [9,10]. The introduction of a lateral voltage has the potential to revolutionize the functional analysis of ion channels as the evolution from diodes (single input) to transistors (double inputs) accelerated the progress in the semiconductor technology [11].
2. BLM-Based Screening Systems for Examining Drug Side Effects and Their Prospect for Personalized Medicine
Ion channel proteins are major targets of drug design for the treatment of various diseases, such as neural disorders, cardiac arrhythmia, cancer, respiratory disorders, and pain [13,14,15,16]. However, unintentional interactions between drugs and ion channels also result in severe side effects. A representative example that received considerable attention is the human ether-a-go-go-related gene (hERG) channel, a voltage-dependent potassium channel in the heart muscle [17]. Diverse groups of drugs have caused an unintentional blockade of the hERG channels, which sometimes induces life-threatening arrhythmias. Many of these drugs have been withdrawn due to their serious side effects on the hERG channel [17,18]. In addition, a possible relationship has been proposed between such drug-induced arrhythmia and the hERG channel genotypes [19].
Another key factor in the BLM-based drug-screening platform is how to consider the effect of oil (nonvolatile organic solvents), such as decane, hexadecane, squalane, and squalene. Since the first report of the formation of a BLM by Mueller et al. in 1962 [20], BLMs have been formed via the self-assembly of amphiphilic lipids in an aqueous environment [6,21,22]. A nonvolatile organic solvent is often effectively utilized to provide an oil–water interface to assist in the formation of a stable BLM [22,23,24,25,26]. However, when applying the BLM systems to a drug screening platform, the presence of oil might cause an increase in the concentration of hydrophobic drugs in the oil and add further complexity to determining the concentration of a target drug in an aqueous phase [6]. Therefore, a solvent-free BLM would be preferable as a drug-screening platform, though the BLM stability could be further weakened in the absence of a solvent.
2.1. Fabrication of Tapered Apertures to Form Stable Solvent-Free BLMs
BLMs are commonly formed either in micro- and nano-apertures that are fabricated in insulating partitions [21,22,24,28,29] or at the interface between water droplets and lipid-containing oils [25,26,27,30]. BLM formation in an aperture still requires oil to seal the gap between the ultrathin BLMs and the partition. If we can reduce the gap and form a smooth connection between the BLM and the partition, it is possible to produce stable BLMs without the need for oil. To achieve this, we adopted the silicon (Si) micro-fabrication technologies to produce circular micro-apertures (φ: 20–60 µm) whose edges were smoothly tapered [12,31,32]. The micro-aperture was fabricated on a silicon nitride (SiN) layer on a Si chip by standard photolithography methods and a wet-etching method. The edge of the fabricated aperture tapered gradually. Solvent-free BLMs were stably formed in the tapered aperture by the folding method. The resulting product had an average lifetime of 16 h [33] and a maximum lifetime of 20 days [12]. In addition to static stability, the BLMs were also mechanically stable, exhibiting tolerance to various mechanical shocks, including repetitive exchanges of the solution, the movement of water around the BLM, and the application of a centrifugal force [12,21,33,34].
2.1. Fabrication of Tapered Apertures to Form Stable Solvent-Free BLMs
To illustrate the potential of using BLMs containing ion channels as a drug-screening platform, we incorporated hERG channels into stable BLM systems. The hERG channel was synthesized using a wheat germ-cell-free expression system and was incorporated into the solvent-free BLMs via fusion between liposomes containing the hERG channel and the BLMs [12]. The incorporation process was facilitated by applying a centrifugal force to cause the proteoliposomes to concentrate near the BLMs [34].
3. Lateral Voltage as a New Input to BLM Systems
Functional evaluations of ion channels are commonly performed based on the current measurement under the control of a transmembrane voltage, the potential difference between the solutions on both sides of the membranes, including both biological cell membranes, and artificially formed BLMs. In both cases, the transmembrane voltage is applied via two electrodes immersed in two buffer solutions that sandwich the membranes. This configuration has not changed substantially since the discovery of the voltage-clamp method, irrespective of extensive progress and changes in target samples from squid giant axons [45,46] to neuronal cells [47], eukaryotic cells that can overexpress the ion channel of interest [48,49], and artificial BLMs containing cell-free synthesized channels [12,30,50,51,52,53].
3.1. Fabrication of Electrode-Wired Membrane Support for the Application of Lateral Voltage
Some scholars first attempted to wire the electrodes on the Si chips [10]. By using the Si chip (h) as the starting material, aluminum (Al) electrodes were deposited on the chip by thermal evaporation. A SiO2 layer was sputtered at the center part of the chip to function as a passivation layer to protect the electrodes from corrosion. Finally, a fluoropolymer CYTOP® was formed on the Si side of the chip.
Teflon films have commonly been used as a support in forming BLMs [22,54,55]. The scholars also used Teflon as a base material for wiring electrodes for lateral voltages. Micro-apertures (φ = 100–150 µm) were first formed on a Teflon film by an electric spark. Titanium (Ti) electrodes were then deposited on the top of the film by electron beam (EB) evaporation (thickness: 200 nm). Similar to the Si chip process, we formed a protective SiO2 layer (thickness: 300 nm) on the Ti electrodes. We then deposited a platinum (Pt) layer on the exposed Ti electrodes to prevent the oxidation of the Ti surfaces. The fabricated Si chip and Teflon film were silanized with (tridecafluoro-1,1,2,2-tetrahydrooctyl) dimethylchlorosilane (PFDS) at room temperature [12,33].
3.2. BLM Formation in Electrode-Wired Membrane Supports
The formation of BLMs was first investigated using the electrode-wired Si chip and the Teflon film. We confirmed that there was no leakage current between the two metal electrodes on the membrane support. The BLM formation was examined by the folding method. In the case of the Teflon film, the aperture was pre-treated with hexadecane to seal the gap between the nm-thick BLM and the μm-thick Teflon film. After folding two lipid monolayers across the apertures fabricated in both types of membrane supports, the resistance between the two Ag/AgCl electrodes was found to exceed 200 GΩ, thus confirming that highly resistive BLMs were formed. No significant changes in the BLM resistance and capacitance were observed between the membrane supports with and without electrode wiring. Therefore, the presence of electrodes and SiO2 layers around the apertures had no measurable effects on the formation of the BLMs.
3.3. Effect of a Lateral Voltage on the Activities of Ion Channels Embedded in BLMs
As described in Section 2, ion channel functions, especially those of voltage-gated channels, have been analyzed in terms of transmembrane voltage [18,56]. For example, the activities of hERG channels are commonly elicited by step pulses of transmembrane voltages (Figure 5b). However, it was observed that some ion channels rapidly lost their activities to show no opening events. After passing through such a non-conducting state, they never returned to show channel activities. If another input, namely a lateral voltage, were to be applied to the BLM system, the lateral voltage may switch the conducting activities of the channel. We examined this possibility by using the human voltage-gated sodium channel (NaV1.5) as an illustrative example. The NaV1.5 channel plays a crucial role in the generation of an action potential in cardiomyocytes [57,58].
The BLMs were formed in Teflon films on which two Ti electrodes were patterned. The proteoliposomes were prepared from NaV1.5-transfected HEK293T cells and were fused to the BLMs for the incorporation of the channel. When a transmembrane voltage protocol that is commonly used to elicit NaV1.5 activities [59] was applied to the BLMs, sporadic channel currents were observed. However, repeated sweeps of the voltage protocol led to a disappearance of channel activities. The channel activities failed to be recovered on the further repetition of the transmembrane voltage protocol. When we additionally applied a lateral voltage (DC 0.5 V), the channel activities were recovered and drastically enhanced. The channel activities under the application of the lateral voltage were similar to that of the initial channel activities. These channel activities were completely blocked by the addition of tetrodotoxin (TTX), a specific inhibitor of sodium channels. No significant current responses were elicited by the same transmembrane protocol when a lateral voltage of DC 4 V was applied to pure BLMs without ion channels. These results suggest that applying a lateral voltage can allow the activities of the NaV1.5 channel that were in non-conductive states to be recovered. The lateral voltage can be a useful additional input for the functional analysis of ion channels, whose activities would have been concealed under the traditional (transmembrane) voltage-clamp conditions.
This entry is adapted from the peer-reviewed paper 10.3390/membranes12090863
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