Membrane Proteins on Artificial Lipid Membranes: History
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Contributor:
Toshiyuki Tosaka
,
Koki Kamiya

Membrane proteins play an important role in key cellular functions, such as signal transduction, apoptosis, and metabolism. Therefore, structural and functional studies of these proteins are essential in fields such as fundamental biology, medical science, pharmacology, biotechnology, and bioengineering.

  • membrane proteins
  • ion channels
  • nanopores
  • giant lipid vesicles
  • liposome
  • proteoliposome
  • planar bilayer lipid membrane
  • artificial cell model

1. Introduction

Membrane proteins are a vital component of the cell membrane, which is composed of a phospholipid bilayer. They play a critical role in various cellular functions, such as molecular transportation, conversion, production, and transduction, including signal transduction, apoptosis, and metabolism [1][2][3][4][5][6][7][8][9][10][11]. Different types of membrane proteins on the cell membrane interact to activate these functions. G protein-coupled receptors (GPCR) and ion channels, which are types of membrane proteins, are important targets for drugs used to treat diseases such as cancer, neurodegenerative disease, metabolic disease and cardiovascular disease [12][13][14][15][16][17]. However, investigating the precise elemental reaction and structure of a membrane protein in complex cell reactions activated by several types of membrane proteins can be challenging. To overcome this issue, methodologies for investigating the function of membrane proteins purified from biological cells have been developed. In the early 1970s, proteoliposomes were first developed by reconstituting the Ca2+-dependent ATPase of the sarcoplasmic reticulum into nano-sized liposomes [18][19]. In the late 1980s, the proteoliposomes with K+-channels of the sarcoplasmic reticulum were generated using the cell-sized liposomes with diameters of approximately 10 μm, due to the measurement of single ion channel current [20]. Recently, cell-sized proteoliposomes with two or three types of membrane proteins were formed to connect each membrane protein reaction [21][22][23]. Purified membrane proteins have been crystallized to reveal their structures, and they have been reconstituted into artificial cell membranes, such as liposomes, planar lipid bilayers, and bicelles, to observe their functions [24][25][26]. These artificial cell membranes consist of phospholipid bilayers, and the structure and function of the membrane proteins reconstituted into them have been identified using various techniques, such as NMR, fluorescence activated cell sorter, cryo-electron microscopy (cryo-EM), fluorescence spectrophotometry, patch clamp method (for nanopores or ion channels), and optical microscopy, including fluorescence microscopy, and confocal microscopy [25][27][28][29][30][31][32][33][34]. These investigations have not only revealed the functions of membrane proteins but also led to the development of biological sensors using membrane proteins because of their highly specific interactions with ligands and membrane proteins [35][36][37]. Thus, studies of membrane proteins can have a wide range of applications (Figure 1).
Figure 1. Schematic illustration of functional studies and applications of membrane proteins on artificial lipid membranes.

2. Formation of Cell-Sized Lipid Vesicles

Membrane proteins are typically reconstituted into artificial cell membranes such as lipid vesicles, liposomes, and bicelles to maintain their structure and function. In this section, the researchers introduce methods for forming lipid vesicles and liposomes, from conventional techniques to recent microfluidic approaches. Liposomes and lipid vesicles, discovered by Bangham in the 1960s, consist of a phospholipid bilayer [38]. Conventional methods for liposome formation include gentle hydration and electroformation [39][40][41][42][43]. With the gentle hydration method, phospholipids dissolved in chloroform are added to a glass microtube, and the films are formed by evaporating the chloroform under flowing argon or nitrogen gas. The lipid films are then hydrated with an aqueous solution (pure water or buffer solution), forming giant liposomes with diameters of 1–100 μm. Nano-sized liposomes are obtained by vortexing or sonication of the lipid films [1]. With the electroformation method, a lipid film is formed on the surface of an indium tin oxide (ITO)-coated glass. An alternating current (AC) electric field is applied to the lipid films containing a hydrated solution (pure water or a buffer solution with low salt concentration) to enhance lipid hydration. Although the giant liposomes formed by the electroformation method have more unilamellar membranes than those formed by the gentle hydration method, the former has lower liposome production than the latter. Methods have been developed to improve the drawbacks of both the gentle hydration and electroformation methods. These conventional liposome formation methods have been used for studying membrane dynamics, functional properties of membrane proteins, and encapsulation of biological reactions.
The gentle hydration and electroformation methods are limited in their ability to produce liposomes with uniform sizes, in their high encapsulation efficiencies, and in their asymmetric lipid distributions. Microfluidic technologies have been integrated into liposome formation to overcome these limitations and achieve a uniform size and high encapsulation efficiency. The droplet emulsion transfer method, developed by Pautot et al. [44][45], is the basis for many of the liposome formation techniques using microfluidic technologies. The liposome formation procedure with the droplet emulsion transfer method is as follows: First, water-in-oil (w/o) emulsions are generated by vortexing or sonication of buffer solution and phospholipid solution dissolved in an organic solvent such as mineral oil or n-decane. The w/o emulsions are then added to the oil phase in a microtube. When the w/o emulsions are transferred to the lipid monolayer between the oil phase and the aqueous phase, liposomes are generated. Asymmetric lipid membranes between the outer leaflet and the inner leaflet can be generated by changing the lipid compositions of the w/o emulsions and the lipid monolayer. By applying the droplet emulsion transfer method, many high-throughput methods for forming liposomes using microfluidic devices have been developed [46][47][48][49][50][51][52][53]. These methods integrate the system of w/o emulsion generation and emulsion transfer from the oil phase to the aqueous phase. Like other methods of liposome formation using microfluidic devices, a new approach has been developed that creates single or multicompartment liposomes by utilizing the de-wetting phenomena of a water-in-oil-in-water double emulsion using a coaxial microcapillary fluidic device [54][55][56][57][58][59][60][61][62].
Another method for forming asymmetric lipid vesicles without an organic solvent (n-decane) between the lipid bilayer is by applying a pulsed jet flow against a planar lipid bilayer in a double-well device (called the pulsed-jet flow method)  [63][64]. The lipid bilayer of liposomes formed by the microfluidic technology method is normally contaminated with an organic solvent, which can cause instability of the liposomes. In contrast, liposomes formed by the pulsed-jet flow method have long-term stability (up to one week). The flip-flop phenomenon and membrane protein reconstitution have been investigated on the asymmetric lipid vesicles formed by this method [64][65]. Modification of the double-well device allows for the formation of nano-sized asymmetric lipid vesicles [66], automatic formation of liposomes [67][68], and the formation of vesicles-in-a-vesicle structures that mimic the asymmetric lipid composition of plasma membranes and intracellular vesicle membranes [69]. Multiple enzyme reactions can be created within the liposomes formed by microfluidic devices [70]. A complex of cell-penetrating peptides and water-soluble proteins can interact with asymmetric lipid vesicles containing negatively charged lipids on the inner leaflet, and the water-soluble proteins can be transferred into the asymmetric lipid vesicles [71].

3. Methodology of Reconstitution of Membrane Proteins into Cell-Sized Artificial Membrane

Giant unilamellar vesicles (GUVs) are widely used as models of artificial cells [72][73]. To understand complex biological phenomena in cells, membrane protein function has been studied by reconstituting membrane proteins into GUVs. Various methods have been developed for this, including rehydration, membrane fusion, detergent removal, and direct insertion (Table 1) [74][75][76][77][78][79][80][81][82][83][84][85][86][87][88][89][90].
Table 1. Selected examples of membrane proteins reconstitution in GUVs.

Protein

Organism

Type

TM

Region

Complex

TM

Number

Method

Membrane Composition

Ref.

BmrC/BmrD

Bacillus subtilis

ABC transporter

α−helix

BmrC/BmrD

12

detergent mediate

reconstitution

DPhPC + DOPC/DOPE

or DOPC/Sph/chol

[74]

BR

Halophilic archaea

proton pump

α−helix

−

7

fusion

EPC:EPA (9:1 [mol])

[74]

CXCR4

Homo sapiens

GPCR

signaling protein

α−helix

homodimer

14 (7 × 2)

fusion

DOPC

[75]

EmrE

Escherichia coli

multidrug transporter

α−helix

homodimer

8 (4 × 2)

direct reconstitution

POPC

[83]

ETB

Homo sapiens

GPCR

signaling protein

α−helix

−

7

fusion

DOPC

[75]

F1F0-ATP

synthase

Escherichia coli

ATP production

α−helix

F1–F0

28

rehydration

E. coli total lipid extract

[84]

FhuA

Escherichia coli

ferrichrome-iron

receptor

β−strand

−

22

fusion

EPC:Tx-DHPE (99.5:0.5)

[74]

GL1

Homo sapiens

γ-aminobutyric

acid receptor

α−helix

−

anchored

rehydration

DOPE:DOPC:DOPE-Atto647

(30:69.5:0.5)

[85]

GLUT1

Homo sapiens

glucose transporter

α−helix

−

12

fusion

DOPC:DMPE-RhB:Biotylated PE

(99.7:0.2:0.1)

[86]

IFITM3

Homo sapiens

enveloped virus

inhibitor

α−helix

−

1

detergent mediate

reconstitution

POPC:cholesterol:Liss-Rho-PE

(99:0.5:0.5 [mol])

[87]

KcsA

Streptomyces lividans

potassium channels

α−helix

homotetramer

8 (2 × 4)

direct reconstitution

DOPG or DOPE

[88]

KvAP

Aeropyrum pernix

voltage-gated

potassium channels

α−helix

heteromer or homotetramer

8 (2 × 4)

rehydration

DPhPC or EPC:EPA (9:1)

[89]

OmpF

Escherichia coli

porin

β−strand

−

16

direct reconstitution

PDMS26-b-PMOXA9

[90]

OmpG

Escherichia coli

porin

β−strand

−

14

direct reconstitution

Outer membrane: DOPC

Inner membrane: oleosin

[76]

OmpLA

Escherichia coli

phospholipase

β−strand

homodimer

24 (12 × 2)

direct reconstitution

DOPC:DOPG (1:3)

[77]

PR

SAR86 group of

marine γ-proteobacteria

proton transport

α−helix

−

7

direct reconstitution

POPC

[78]

RC

Rhodobacter sphaeroides

electron transport

α−helix

−

10

detergent mediate

reconstitution

POPC:POPG (9:1 [mol])

[79]

SLO

Streptococcus pyogenes

toxin

α−helix

homo

36~40 mer

36~40

rehydration

POPC, DOPC, SOPC, POPG

[80]

TmrAB

thermus thermophilus

ABC transporter

α−helix

−

6 × 2

rehydration

POPC:POPG:POPE:

biotinylated-DOPE

(40:30:29:1 [mol])

[81]

TolC

Escherichia coli

expulsion of diverse

molecules from the cell

β−strand

homotrimer

12 (4 × 3)

rehydration

DOPC:DOPS:Atto647-DOPE

(91.2:8:0.8)

[85]

t-SNARE

Homo sapiens

endocytosis/exocytosis

α−helix

Syntaxin

-SNAP25

1

rehydration

DOPC:DOPS:Atto647-DOPE

(91.2:8:0.8)

[85]

VDAC1

Homo sapiens

voltage dependent

anion channel

β−strand

−

19

fusion

soybean polar extract:

cholesterol (9:1)

[82]

4. Single-Channel Recording Using Planar Bilayer Lipid Membrane Systems

The function of membrane proteins on cell membranes, such as ion channels and transporters, has been studied using the patch-clamp method. Neher and Sakmann [91] developed the patch-clamp method to measure the function of ion channels and transporters at the single molecule level and in real time. While various patch-clamp methods have been developed for measuring ion currents of ion channels in biological target membranes, artificial planar bilayer lipid membrane (BLM) systems have recently been developed for an engineering-oriented approach [92]. In BLM systems, the target ion channels purified from living cells are reconstituted in the BLM, and the ionic current of the ion channels is measured using a patch clamp amplifier. BLMs allow us to perform functional studies of ion channels in a well-defined environment, such as buffer composition, lipid composition, inhibitor or activator concentration, and membrane potential.
The BLMs (Figure 2a–c) can also be formed by the painting method [93], the Langmuir–Blodgett method [94], or the droplet contact method [63]. In the painting method, a lipid solution (phospholipids dissolved in an organic solvent) is applied across a small aperture that separates two aqueous compartments, forming lipid monolayers. Then, a BLM is formed by the contact of each monolayer after the removal of the organic solvent [93]. This method of BLM formation has improved the Langmuir–Blodgett method by containing a smaller amount of an organic solvent because the presence of the organic solvent in the BLM causes denaturation of the membrane proteins. In the Langmuir–Blodgett method, BLMs are prepared by the apposition of two lipid monolayers spread at the air/water interface. The two monolayers are combined in a small aperture of a Teflon film with a thin layer of n-hexadecane or squalene [94]. In the droplet contact method, the lipid bilayer is formed by contacting two water droplets surrounding the lipid monolayer in the double well chip [63]. The painting method or the Langmuir–Blodgett method suffers from fragility and low reproducibility. Therefore, the use of high-throughput systems for pharmaceutical screenings of the BLM formed by these methods is difficult. Conversely, BLMs formed by the droplet contact method can be easily formed by simply dropping each of the solutions.
Figure 2. Illustration of BLM formation and the reconstituted ion channel. (a) Painting method. (b) Langmuir-Blodgett method. (c) Droplet contact method. (d) Vesicle Fusion method. (e) Nystatin/Ergosterol method.

5. Lipid Vesicles Containing Some Types of Membrane Proteins for Creating Complex Artificial Cell Models

To create artificial cell models that emulate the multiple functions of living cells, various membrane protein functions, such as molecular transportation, molecular conversion, and molecular production, must be reconstituted into lipid vesicles. Additionally, fluorescent probes or biomolecules such as DNA, water-soluble proteins, in vitro transcription-translation systems, and ions have been encapsulated into lipid vesicles. These complex artificial cell models, based on a bottom-up approach, have been constructed worldwide. In this section, the researchers introduce lipid vesicles containing two or more types of membrane proteins.
Ishmukhametov et al. [21] developed lipid vesicles reconstituted with ATP synthase and bo3-oxidase through fusion between cationic and anionic lipid vesicles containing either ATP-synthase or bo3-oxidase. This created a molecular bio-synthetic network linking the primary (proton motive force, PMF) and secondary ATP forms of biological free energy. Detergent removal using a Sephadex G-50 column reconstituted ATP synthase and bo3-oxidase into the lipid vesicles. When reduced dithiothreitol and Coenzyme Q1 were added to the outer solution of the lipid vesicles, PMF was generated toward the inner solution because protons were also transferred to the inner solution. With PMF and KPi added to the inner solution, ATP was synthesized from ADP and pyrophosphate by ATP synthase in the outer solution. The ATP production of this molecular biosynthetic network was detected by luminescence using the luciferin-luciferase system.
Berhanu et al. [22] developed nano-sized lipid vesicles containing ATP synthase and BR for synthesizing proteins of an in vitro transcription–translation system using ATP produced from the nano-sized lipid vesicles by light. Detergent removal using bio-beads reconstituted ATP synthase and BR into the nano-sized lipid vesicles, which were then encapsulated into cell-sized lipid vesicles along with the in vitro transcription–translation system. When light was irradiated to the cell-sized lipid vesicles, BR on the nano-sized lipid vesicle membranes transported protons into the nano-sized lipid vesicles. By increasing the proton concentration into the inner solution of the nano-sized lipid vesicles, ATP-synthase converted ADP to ATP in the outer solution of the nano-sized lipid vesicles (in other words, into the inner solution of the cell-sized lipid vesicles). The produced ATP was consumed as substrates for messenger RNA, energy for phosphorylation of guanosine diphosphate, or energy for aminoacylation of transfer RNA.
Lee et al. [23] developed nano-sized lipid vesicles containing ATP synthase, PR, and plant-derived photosystem II (PSII) for controlling ATP production by changing the proton concentration and switching PR or PSII activity using red or green light. ATP synthase, PR, and PSII were reconstituted in the nano-sized lipid vesicles using a detergent-mediated method. The activation mechanism of the nano-sized lipid vesicles containing ATP-synthase, PR, and PSII was as follows: red light irradiated to the nano-sized lipid vesicles generated protons into the inner solution of the nano-sized lipid vesicles using PSII, which subsequently increased the proton concentration and caused ATP synthase to convert ADP to ATP in the outer solution. Conversely, green light caused protons to move from the inner to the outer solution of the nano-sized lipid vesicles, decreasing the proton concentration and halting ATP synthesis. Cell-sized lipid vesicles containing three types of membrane protein-reconstituted nano-sized lipid vesicles, ionophore, and actin experienced actin polymerizations and morphological changes depending on the ATP concentration generated from the nano-sized lipid vesicles.

This entry is adapted from the peer-reviewed paper 10.3390/ijms24087231

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