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Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Biology

Giant Liposome Preparation for Imaging and Patch-Clamp Electrophysiology

Published: June 21st, 2013

DOI:

10.3791/50227

1Department of Physiology and Biophysics, University of Washington

Reconstituting functional membrane proteins into giant liposomes of defined composition is a powerful approach when combined with patch-clamp electrophysiology. However, conventional giant liposome production may be incompatible with protein stability. We describe protocols for producing giant liposomes from pure lipids or small liposomes containing ion channels.

The reconstitution of ion channels into chemically defined lipid membranes for electrophysiological recording has been a powerful technique to identify and explore the function of these important proteins. However, classical preparations, such as planar bilayers, limit the manipulations and experiments that can be performed on the reconstituted channel and its membrane environment. The more cell-like structure of giant liposomes permits traditional patch-clamp experiments without sacrificing control of the lipid environment.

Electroformation is an efficient mean to produce giant liposomes >10 μm in diameter which relies on the application of alternating voltage to a thin, ordered lipid film deposited on an electrode surface. However, since the classical protocol calls for the lipids to be deposited from organic solvents, it is not compatible with less robust membrane proteins like ion channels and must be modified. Recently, protocols have been developed to electroform giant liposomes from partially dehydrated small liposomes, which we have adapted to protein-containing liposomes in our laboratory.

We present here the background, equipment, techniques, and pitfalls of electroformation of giant liposomes from small liposome dispersions. We begin with the classic protocol, which should be mastered first before attempting the more challenging protocols that follow. We demonstrate the process of controlled partial dehydration of small liposomes using vapor equilibrium with saturated salt solutions. Finally, we demonstrate the process of electroformation itself. We will describe simple, inexpensive equipment that can be made in-house to produce high-quality liposomes, and describe visual inspection of the preparation at each stage to ensure the best results.

Giant liposomes (often called giant unilamellar vesicles, or GUVs) have primarily been used to study the physics and physical chemistry of lipid bilayers, including studies of bilayer deformation, lateral phase coexistence ("rafts"), membrane fusion, etc1-4. They have a grossly cell-like structure: spherical shell of membrane surrounding an aqueous interior which can easily be made different than the surrounding aqueous buffer. They are, by definition, ≈1-100 μm in diameter, so they can be imaged using a variety of light microscopy approaches. They can be made taut using osmotic gradients or mechanically applied tension, so that while gene....

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1. Deposition of Lipids from Organic Solvents: Classical Protocol

  1. Remove lipids from storage at -20 °C or -80 °C; warm to RT. Caution: lipids are extremely hygroscopic, and many are sensitive to oxygen. Cover lipids in dry Argon or Nitrogen gas and in all steps minimize exposure to air.
  2. If necessary, suspend the lipids in chloroform or cyclohexane at 1-10 mg/ml; note that manufacturer's stated concentrations are typically nominal only. CAUTION: Wear appropriate .......

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In our examples, we prepare liposomes from a mixture of approximately 55 mol% POPC (1-palmitoyl-2-oleoyl-sn-glycero-phosphocholine), 15 mol% POPS (1-palmitoyl-2-oleoyl-sn-glycero-phosphoserine, 30 mol% cholesterol, and 0.1 mol% Texas Red-labeled 1,2-dipalmitoyl-sn-phosphoethanolamine (TxR-DPPE). This composition was chosen as approximately representative of dorsal root ganglion lipids18. We note that 15 mol% charged lipid (here POPS) is near the limit of what can be used in electrofo.......

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Electroformation of giant liposomes has developed into a flexible technique compatible with diverse lipids, preparations, and buffers. Careful control of the lipid deposition process is most critical to success. We have presented simple tools to make controlled deposition of lipids from small liposome preparations a straightforward process. The relative humidity is critical to proper dehydration of the initial liposomes, and the optimum value will vary with the initial concentration of solutes in the liposome suspension........

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We thank Bryan Venema and Eric Martinson for constructing the electroformation apparatus. This work was funded by grants from the National Institutes of General Medical Sciences of the National Institutes of Health (R01GM100718 to SEG) and the National Eye Institute of the National Institutes of Health (R01EY017564 to SEG).

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Name Company Catalog Number Comments
Name of the reagent Company Catalogue number Comments (optional)
Digital Multimeter Agilent Technologies, www.agilent.com U1232A or similar Any multimeter will do, but avoid old style analog ohmmeters which apply much more current to the resistance under test.
  Fluke 117 or 177 Any multimeter will do, but avoid old style analog ohmmeters which apply much more current to the resistance under test.
Function Generator Agilent Technologies, www.agilent.com 33210A or similar Most function generators work for simple protocols. This programmable model is useful for advanced electroformation protocols. Make sure the generator can drive 10 V peak-to-peak into a 50 Ω load
ITO coated glass slides Delta Technologies, Loveland, CO www.delta-technologies.com CB-90IN-S107 or similar Break these in half to make two slides, 25 mm x 37 mm
Temperature controller Omega Engineering Stamford, CT www.omega.com CNi3233 or similar  
Hygrometer Extech, Nashua, NH, www.extech.com 445815  
Silicone rubber sheet McMaster-Carr Elmhurst, IL www.mcmaster.com 87315K64 Use USP Grade VI silicone for its high purity
EMI gasket Laird Technologies www.lairdtech.com 4202-PA-51H-01800 or similar Distributed by Mouser www.mouser.com
TxR-DHPE Life Technologies, Carlsbad, CA www.lifetechnologies.com T1395MP Other fluorescently labeled lipids are available, but TxR-DHPE is one of the brightest and most photostable.
POPC Avanti Polar Lipids, Alabaster, AL www.avantilipids.com 850457P or 850457C Lipids can be ordered as powders (P) or in chloroform (C)
POPS Avanti Polar Lipids 840034P/C  
Cholesterol Sigma-Aldrich C8667  

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