Giant Liposome Preparation for Imaging and Patch-Clamp Electrophysiology

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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, etc^1-4. 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 generally soft, their properties can be manipulated for easy handling. In particular, controlling the "stiffness" of the liposome makes it straightforward to form "liposome-attached" or excised patches for electrophysiology. In the past, ion channel reconstitution was largely performed in planar lipid bilayers. Now, the ability to form patches from giant liposomes and use the considerable quiver of tools developed for conventional electrophysiology (fluorescence microscopy, micropipette aspiration, rapid perfusion and temperature control, etc.) makes giant liposomes increasingly attractive for reconstitution studies^5,6.

Giant liposomes have been made by many strategies. In fact, giant liposomes form spontaneously by a swelling process when a dried lipid film is rehydrated^4,7,8. The desire to more rapidly prepare larger, more uniform liposomes led researchers to other approaches, chief among them electroformation^1,9. Electroformation also relies on hydration of a dried lipid film, but speeds the process through the application of an oscillating electric field across the lipid film. The field is applied through two electrodes, either platinum wires or Indium-Tin-Oxide (ITO) coated glass slides, separated by water or buffer and onto which the lipids are deposited. By speeding the swelling of liposomes, one achieves a higher yield of larger liposomes. Thus, electroformation has become the default method to produce giant liposomes⁴.

The mechanism of electroformation is not fully understood, and most of the protocols are developed empirically (e.g. ^10,11). Nonetheless, we can learn a little about what to expect by considering the theory and some empirical results. It is widely believed that electroformation occurs by driving electro-osmotic flow of buffer between individual lipid bilayers stacked in the deposited lipid film^10,11. Electrostatic coupling to thermal fluctuations of the lipid bilayers is probably also involved¹². These hypotheses qualitatively predict upper limits for the electric field frequency and strength that can be used^10,12. In particular, it is predicted that high conductivity solutions (i.e. physiological salt solutions) reduce the electrohydrodynamic forces that may initiate the liposome electroformation¹². Electroosmotic flow rates generally decrease with increasing salt concentration and are frequently peaked at some electric field oscillation frequency (e.g. albeit in a different geometry, Green et al.¹³). Thus, higher field strengths and higher frequencies are reasonable for high conductivity solutions, within limits¹⁰.

However, membrane proteins are likely to be incompatible with the usual method of depositing lipids onto electrodes for the electroswelling procedure, namely in organic solvents which are then evaporated off to leave a thin lipid film. There are two principal paths around this difficulty: to incorporate proteins after giant liposome formation, or to adapt how the lipids are deposited. Our approach builds on others^5,11 to deposit the lipids and reconstituted membrane protein together from a suspension of small or large "proteoliposomes". We describe the lengthy and more challenging process of producing proteoliposomes from purified protein and lipids elsewhere (Collins and Gordon, in review). Here we describe the protocol in the absence of any protein, but it is the same when protein is incorporated; we include results showing that proteoliposomes containing the ion channel TRPV1 can be transformed into GUVs and used for patch-clamp electrophysiology. In any electroformation approach, visual inspection of the lipid sample during the lipid deposition process is critical to success.

Our approach may be relevant beyond the specialized application to ion channel reconstitution. In the time since we first developed this protocol and now, it has also been shown how the way in which lipids are deposited on electrodes for electroformation affects the compositional heterogeneity of the resulting GUVs. Baykal-Caglar et al.¹⁴ showed that GUVs formed from carefully dehydrated liposomes had a 2.5 times smaller variation in the miscibility transition temperature of GUVs formed from mixtures of various phospholipids and cholesterol. Their work indicates that lipids, and especially cholesterol, may precipitate from the lipid mixture when deposited from organic solvents, resulting in large spatial variation in composition of the deposited lipid film. This is especially important for studies of lipid membrane phase behavior, but may also be critical to quantitative experiments on ion channel function. Baykal-Caglar et al.'s protocol is similar but not identical to our own, and readers are encouraged to study it as well.

This protocol (see overview, Figure 1) is one of many that could be used. In principle electroformation success depends on the lipid mixture, hydration, temperature, other solutes (especially ions), and of course the voltage and frequency used in formation. As electroformation becomes better understood, we expect to refine our protocol more.

Finally, there is often a steep learning curve in electroforming giant liposomes. We suggest mastering the conventional protocol (Sections 1 and 4, and, if necessary, Section 5) before learning to deposit lipids from liposomal suspensions (Sections 2-5).

Protokół

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 gloves and other personal protective equipment when using organic solvents. Remove the PPE quickly if solvent is spilled on them, as most materials are still permeable to these solvents and they only provide temporary protection.
3. Clean both sides of two 25 x 37.5 mm ITO coated glass slides with ethanol
4. Mix the lipids to achieve the desired molar ratios. For each 1 cm² of area of ITO slides to be coated with lipid, mix ~15-20 μg of lipids. For example, if both 25 x 37.5 mm slides were to be fully coated, we would typically use 30 μl of 10 mg/ml lipid mixture. Note: add 0.1 mol% Texas Red-DPPE for fluorescent imaging, and see below for cautions about fluorescent dyes.
5. Verify that the ITO coated side of the glass slides is facing up by measuring the surface resistance with an ohmmeter or multimeter.
6. Aspirate the lipids into a solvent-resistant syringe. Caution: No glues or plastics, other than PTFE, should contact the organic solvent.
7. With the syringe needle not quite touching the ITO surface, slowly apply the lipid moving the needle back and forth across the slide. Cover the surface as evenly, looking for a "rainbow sheen" on the glass surface.
8. Place the slides quickly under < 1 Torr (1 mmHg) vacuum for 0.5-1 hr to remove any trace solvent. Release vacuum with inert gas.
9. Apply a silicone gasket, with a thin layer of silicone vacuum grease on both sides. Leave at least 5 mm of uncovered slide exposed at one end of the slide.
10. Proceed immediately to Section 4.

2. Small Liposome Preparation for Controlled Dehydration

1. Prepare the lipid mixture as in Steps 1.1 to 1.4.
2. Dry the lipids using a stream of Argon or Nitrogen gas in a 10-15 mm diameter culture tube. For small quantities of lipids, ≤0.5 mg, the resulting film can be hydrated directly after placing it under vacuum for 0.5-1 hr to remove residual solvent; proceed to step 2.5. Larger amounts of lipid tend to trap organic solvents in a thick gel, even under vacuum, and are better prepared by lyophilization; see steps 2.3-4.
3. Suspend the dried lipid film in cyclohexane, cover with Argon and seal with a stopper, place the tube in a cold block and freeze at -80 °C for 1 hr minimum.
4. Place the cold block and lipid sample in a vacuum system capable of reaching 100 mTorr. Apply vacuum; it will "stall" at about 1 Torr as solvent sublimes off. Once solvent is completely removed (~1 hr) vacuum will drop below this level. Release vacuum with inert gas and seal the tube or hydrate immediately.
5. While the lipids are under vacuum, degas hydration buffer using vacuum and sparging^15-17.
6. Hydrate the lipids using degassed buffer at final concentration 1-10 mg/ml. Allow the lipids to hydrate slowly. After 30 min-1 hr, vortex to break up remaining clumps. Wait an additional 0.5-1 hr to allow for complete hydration. Use an osmoticant, such as sorbitol or sucrose up to 200 mM, to prevent complete dehydration in steps below.
7. Prepare 100-200 nm diameter liposomes by extrusion. See manufacturer's extrusion protocol for details. Note that the buffer must have less than ~25 mM ionic strength, or special electroformation voltage protocols will be needed in Section 4.

3. Deposition of Lipids by Controlled Dehydration of Small Liposomes

1. Caution: Our protocol requires lipids to be placed in vapor equilibrium with saturated salt solutions for many hours. We strongly recommend performing the protocol in an inert gas atmosphere, using a glove box or similar enclosure.
2. Caution: If preparing samples containing proteins, avoid complete dehydration. Hydrate the atmosphere in any enclosure with a beaker of warm water, or a sonicator based humidifier. Use a hygrometer to ensure there is at least 30% relative humidity. Use sorbitol or sucrose in the small liposome buffer as an additional precaution to prevent total dehydration.
3. Prepare a saturated salt solution of appropriate relative humidity. Target humidity depends on the osmolarity of the vesicle preparation. For preparations with physiological osmolarity, use 30-45% RH, while low osmolarity vesicle preparations can be adequately dehydrated in equilibrium with 75-90% RH (See also Table 1).
4. Put the saturated salt solution and excess salt in a tightly sealable container with an interior shelf. Commercial food containers for yogurt and granola work very well. Replace the shelf after filling, and make sure fluid is 5-10 mm below the shelf.
5. Clean ITO coated slides as in Section 1. Use the multimeter to determine which side is conductive, and label the non-conductive side with the sample name.
6. Lightly grease both sides of the silicone (USP Grade VI) gasket(s) with one or multiple holes.
7. Place the slides conductive side up on the bench, and apply the silicone gasket(s) to each slide to which lipid will be applied, making sure there is at least 5 mm of exposed slide at one end to connect to the electroformation apparatus. Smooth the gasket to ensure a good seal.
8. Dilute the vesicle preparation in a low-salt isosmotic buffer to ~1-2 mg/ml. We find that higher concentrations produce poor results.
9. Apply lipid to the slide in 1-10 μl drops. Smaller drops generally produce better results.
10. Place the slide on the interior shelf above the saturated salt solution and seal the container tightly. Leave at RT for 3 hr to O/N. The container can be placed at 4 °C, but note that for some salts, the RH depends strongly on temperature, and that cold will increase equilibration time.
11. The resulting film may have a slight rainbow sheen to it, but in any case should appear nearly desiccated. Highly osmotic starting solutions may be impossible to dry completely to a lipid film; this will not adversely affect the results.

4. Electroformation of Giant Liposomes

1. Lipid films, especially those formed from dehydrated liposomes, are easily dislodged. Carefully hydrate each well by placing a 27 G syringe needle at the edge of the gasket and slowly applying buffer. Buffers can include water, ≤200 mM sucrose, ≤1 M sorbitol and ≤5 mM HEPES. More than 10 mM salt solution may require alternate electroformation voltage protocols. Overfill each gasket well by ~10%.
2. Note: once the lipids are hydrated, proceed quickly through the remaining steps, since lipid films begin to delaminate immediately. Yield and size are maximized if electroformation begins promptly.
3. In one smooth motion, apply a second ITO slide, conductive face in, to the top of the gasket. Make sure to have at least 5 mm of overhang outside the gasket area and opposite the first overhang. Press gently to ensure a good seal.
4. Clean the two overhangs using ethanol and verify that sides facing the gasket are conductive with your multimeter.
5. The chamber can be further secured with parafilm or light tension spring clips if desired.
6. Connect the electroformation chamber to a voltage source by securing aluminum or copper bars, "EMI gasket foam", or conductive-adhesive tape to the conductive surfaces of the two slides. We use EMI gasket foam. Plans for a jig and clamp are shown in Figure 5.
7. Verify that there is no electrical short between the contacts, and that the contacts connect properly to the ITO surfaces, using the multimeter.
8. Heat the chamber 10 °C above the highest melting temperature of any of the lipids present; e.g. for DPPC, heat to 52 °C minimum, 10 °C above the chain melting temperature of 42 °C.
9. For low-salt buffers, apply a 10 Hz sine wave, ~0.7 V_rms for each millimeter gap between the two ITO coated surfaces, for 60-90 min. For high salt buffers, other voltage protocols should be used (see references).

5. Imaging and Troubleshooting

1. Image the liposome using an inverted microscope equipped with a filter cube for Rhodamine or Texas Red dyes. The liposome should be spherical, predominantly unilamellar by eye, and free of defects such as "strings" hanging off the liposome.
2. If the liposomes are too small, use less lipid.
3. If there are many defects or few liposomes, this may be due to gel-phase lipids. Consider raising the electroformation temperature.
4. If few or no giant liposomes formed at all from dehydrated small liposomes, reduce the osmotic strength of the liposome buffer, or increase the osmotic strength of the electroformation buffer. An inrush of buffer into the concentrated interstitial buffer solution can cause delamination of the deposited lipid film.
5. If vesicle yield, quality, or size is poor, and you included salts or pH buffers in the electroformation or liposome buffers, consider alternative electroformation voltage protocols. For instance, Pott, et al.¹¹, recommend a three step protocol using a 500 Hz sinewave, raising the voltage from 50-1,300 Vpp/m over 30 min, holding for 90 min, then decreasing the frequency to 50 Hz over 30-60 min. Platinum or titanium electrodes may be needed in this case, but this will not substantially alter the protocol.

Wyniki

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...

Dyskusje

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....

Materiały

Name	Company	Catalog Number	Comments
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