Name: reconstitution of a transmembrane protein, the voltage-gated ion channel, kvap, into giant unilamellar vesicles for microscopy and patch clamp studies
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Description: Watch this Scientific Journal Video about Reconstitution of a Transmembrane Protein, the Voltage-gated Ion Channel, KvAP, into Giant Unilamellar Vesicles for Microscopy and Patch Clamp Studies at JoVE

We thank Susanne Fenz for discussing the possibility of reconstituting proteins by agarose swelling, Feng Ching Tsai for current measurements, and present and former members of the Bassereau group for support and assistance. The project was funded by the Agence Nationale de la Recherche (grant BLAN-0057-01), by the European Commission (NoE SoftComp), by the Universit&#233; Pierre et Marie Curie (grant from the FED21, Dynamique des Syst&#232;mes Complexes). M.G. was supported by an Institut Curie International PhD Fellowship, S.A. by a fellowship from the Fondation pour la Recherche M&#233;dicale, G.E.S.T. by a Marie Curie Incoming International Fellowship from the European Commission and a grant from the Universit&#233; Pierre et Marie Curie. The publication fees were covered by the Labex &#8216;CelTisPhyBio&#8217; (ANR-11-LABX0038).

1. Solution Preparation
<ol>
	<li>Prepare 5 ml of &#8216;SUV buffer&#8217; containing 5 mM KCl, 1 mM HEPES (pH 7.4) or TRIS (pH 7.5), and 2 mM trehalose. Filter the buffer with a 0.2 &#181;m syringe filter and divide into 1 ml aliquots which can be stored at -20 &#176;C. 
	NOTE: Additional details for reagents and instruments are given in the materials list.</li>
	<li>Prepare 40 ml of GUV &#8216;Growth Buffer&#8217; that will fill the GUV interior during film rehydration. For a &#8216;low salt&#8217; growth, combi...

<ol>
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Biomimetic model systems are an important tool for studying the properties and interactions of proteins and membranes. Compared to other reconstituted systems like BLMs or supported lipid membranes, GUV based systems present several opportunities including considerable control of membrane composition, tension and geometry, as well as being truly oil-free. However, incorporating transmembrane proteins, such as KvAP, into GUVs requires significant adaptations of conventional protocols for lipid-only GUVs. The electroformat...

When studying the physical principles that govern living systems, bottom-up approaches allow an experimentalist to control system composition and other parameters that are not easily manipulated in cell-based systems1. For membrane-based processes, Giant Unilamellar Vesicles (GUVs, diameter ~ 1&#8211;100 &#181;m) have proven to be a very useful biomimetic system2&#8211;7 as they are well suited for microscopy studies and micromanipulation8&#8211;10. While there are many different protocols to produce GUVs, most fall into two categories &#8212; emulsion based approaches11,12 and techniques based on rehydrating a lipid film13&#8211;16. In emulsion-based methods, the inner and outer leaflets of the GUV membranes are assembled sequentially from lipid monolayers at water/oil interfaces. This approach is ideal for encapsulating soluble proteins within the GUVs, and for forming GUVs with asymmetric leaflet lipid composition. However, GUVs formed from emulsions can retain traces of solvent that change the membrane&#8217;s mechanical properties17, and the approach is not especially well-suited to trans-membrane protein reconstitution.
Film rehydration methods rely on the fact that drying (dehydration) causes many lipid mixtures to form a multi-lamellar stack of membranes. If this stack is then placed in contact with an aqueous buffer, membranes in the stack will move apart as solvent flows between them and at the surface of the stack, individual membranes can detach to form GUVs13,18 (as well a veritable zoo of other lipidic objects). However, even for optimal buffer and lipid compositions, this classical &#8220;spontaneous swelling&#8221; method has a relatively low yield of defect-free GUVs. One widely used method to boost the yield of defect-free GUVs is &#8220;electroformation&#8221;, in which an alternating current (AC) field is applied during film rehydration. While the mechanism remains poorly understood, &#8220;electroformation&#8221; can give spectacular GUV yields (&#62; 90% in favorable circumstances) for low salt concentration buffers (&#60; 5 mM)14,19, and can even work in physiological buffers (~100 mM) using a higher frequency (500 Hz versus 10 Hz) AC field and platinum electrodes15. An alternative approach to boost the yield of defect-free GUVs is &#8220;gel-assisted swelling&#8221;, in which the lipid solution is deposited onto a polymeric gel substrate rather than the passive (e.g., glass, PTFE) substrates used in classical &#8220;spontaneous swelling&#8221;. When the resultant lipid/gel film is rehydrated, GUVs can rapidly form even for physiological buffers16,20.
All these methods can produce lipid-only GUVs which can be used to study membrane associated phenomena such as the interaction between soluble proteins and membranes. However, to incorporate a trans-membrane protein into GUVs, significant modifications are needed to ensure that the protein remains in a functional state throughout the reconstitution procedure. While solutions of lipids in organic solvents (e.g., chloroform, cyclohexane) are ideal for producing lipid films, trans-membrane proteins are typically only stable when their hydrophobic trans-membrane domain is embedded in a lipid bilayer, or surrounded by a detergent micelle (e.g., during protein purification). Thus, the starting material for a reconstitution is typically native membranes, purified protein in a detergent solution, or small unilamellar protein-containing vesicles (proteo-SUVs) and/or multi-lamellar vesicles (proteo-MLVs) formed by detergent removal in the presence of lipids. Most methods to incorporate these membrane proteins into GUVs fall into three categories.
Direct Insertion: Trans-membrane protein suspended in detergent is mixed with pre-formed, lipid-only, mildly detergent solubilized GUVs, and the detergent then removed using biobeads21. While conceptually simple, this method requires precise control of the detergent concentration, as too high a detergent concentration can dissolve the GUVs while too low a concentration can cause the protein to unfold or aggregate.
GUV/Proteo-SUV Fusion: Protein in proteo-SUVs is combined with pre-formed, lipid-only GUVs and fusion is facilitated with special fusogenic peptides22 or detergent21. Typically the extent of fusion is limited leading to GUVs with low protein density.
Dehydration/Rehydration: A protein-containing lipid film is formed by partial dehydration of a proteo-SUV (or proteo-MLV) solution and GUVs are then grown as for a pure lipid film. The obvious challenge is to protect the protein during the partial dehydration step23, but the method has been successfully used to reconstitute trans-membrane proteins such as Bacteriorhodopsin, Calcium-ATPase, Integrin and VDAC into GUVs7,23&#8211;25.
This article describes dehydration/rehydration protocols to make GUVs containing the voltage-gated potassium channel, KvAP, from the hyper-thermophilic Archaea, Aeropyrum pernix. KvAP has a high degree of homology to eukaryotic voltage dependent potassium channels26 and a known crystal structure27, making it a good model for studying the mechanism of voltage gating. Production of the proteo-SUVs has been described in detail previously and is not part of this tutorial26,28,29. Importantly, KvAP proteo-SUVs do not have to be produced for each GUV preparation, as they can be stored in small (e.g., 10 &#181;l) aliquots at -80 &#176;C for extended periods of time (&#62; 1 year). Electroformation or gel-assisted swelling can then be used to grow GUVs from the KvAP proteo-SUVs (or proteo-MLVs).
The key steps for the electroformation protocol are illustrated in Figure 1. Droplets of a solution of SUVs containing the protein are deposited on platinum wires (shown in Figure 2). Partial dehydration of the SUV suspension leads to the formation of a lipid protein film through the fusion of SUVs. During rehydration, an AC field is applied to the electrodes to assist the lipid layers to delaminate and form GUVs. A 10 Hz field works well when using &#8220;low-salt&#8221; (&#60; 5 mM) rehydration buffer28 and GUVs take several hours to grow. In contrast, physiological buffers (containing ~100 mM salt) work well with a lower voltage, 500 Hz AC field but require a prolonged (~12 hr) swelling period15. This method is based upon an earlier protocol using ITO slides24, but uses a custom chamber containing two platinum wires as shown in Figure 2 (see the discussion for design details and suggestions for simpler, improvised chambers).
Figure 3 illustrates the gel-assisted swelling method. The protocol works well with buffers with physiological salt concentrations, is rapid, and produces GUVs with a more homogeneous protein distribution. However, the yield of isolated, apparently defect-free GUVs (i.e., the GUV membrane is uniform at optical length-scales and does not enclose any objects) is lower, although it provides a sufficient number for patch-clamp and micro-manipulation experiments. This method was based on a protocol using agarose gel to produce lipid-only GUVs16 and requires less specialized equipment than the electroformation method.
The characterization of GUVs with fluorescence microscopy is described, as well as procedures using a standard patch-clamp set-up to measure KvAP activity in &#8220;inside-out&#8221; excised membrane patches.
Growing protein-containing GUVs can be more difficult than lipid-only GUVs. In particular, the final GUV yield can depend sensitively on exactly how the SUV solution is deposited and dehydrated to form the membrane stack. For someone without any previous experience with GUVs, it may be helpful to first grow lipid-only GUVs following a conventional protocol15,16 in which the membrane film is formed by depositing lipids from an organic solvent. Once the conventional protocol works well, SUV deposition and partial dehydration can then be mastered using lipid-only SUVs, which are also very helpful when adjusting the protocol for a new lipid composition. When GUVs grow reliably from lipid-only SUVs, it is then only a small step to produce protein-containing GUVs from proteo-SUVs.

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			<td style="width:132px;">Avanti Polar Lipids&#160;</td>
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			<td style="width:132px;">Avanti Polar Lipids&#160;</td>
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			<td style="width:149px;"></td>
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		<tr height="60">
			<td dir="LTR" height="60" style="height:60px;width:163px;">18:1 (&#916;9-Cis) PC (DOPC) 1,2-dioleoyl-sn-glycero-3-phosphocholine</td>
			<td style="width:132px;">Avanti Polar Lipids&#160;</td>
			<td>850375P</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="40">
			<td dir="LTR" height="40" style="height:40px;width:163px;">cholesterol (ovine wool, &#62;98%)&#160;</td>
			<td style="width:132px;">Avanti Polar Lipids&#160;</td>
			<td>700000P</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:163px;">TRed-DHPE</td>
			<td style="width:132px;">Invirtogen</td>
			<td>T-1395MP</td>
			<td style="width:149px;">labeled lipid</td>
		</tr>
		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:163px;">BPTR-Ceramide</td>
			<td style="width:132px;">Invirtogen</td>
			<td>D-7540&#160;</td>
			<td style="width:149px;">labeled lipid</td>
		</tr>
		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:163px;">Choloroform</td>
			<td style="width:132px;">VWR</td>
			<td>22711.290</td>
			<td style="width:149px;">AnalaR Normapur</td>
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			<td dir="LTR" height="20" style="height:20px;width:163px;">Acetone</td>
			<td style="width:132px;">VWR</td>
			<td>20066.296</td>
			<td style="width:149px;">AnalaR Normapur</td>
		</tr>
		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:163px;">Ethanol</td>
			<td style="width:132px;">VWR</td>
			<td>20821.330</td>
			<td style="width:149px;">AnalaR Normapur</td>
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		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:163px;">Kimwipe</td>
			<td style="width:132px;">Kimtech</td>
			<td>7552</td>
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		<tr height="40">
			<td dir="LTR" height="40" style="height:40px;width:163px;">Hamilton syringes&#160;</td>
			<td style="width:132px;">Hamilton Bonaduz AG</td>
			<td></td>
			<td style="width:149px;">diverse</td>
		</tr>
		<tr height="40">
			<td dir="LTR" height="40" style="height:40px;width:163px;">Amber vials and teflon cups</td>
			<td style="width:132px;">Sigma</td>
			<td>SU860083 and SU860076</td>
			<td style="width:149px;"></td>
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		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:163px;">Parafilm</td>
			<td style="width:132px;">VWR</td>
			<td>291-1214</td>
			<td style="width:149px;"></td>
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		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:163px;">microcentrifuge tube</td>
			<td style="width:132px;">eppendorf</td>
			<td></td>
			<td style="width:149px;">diverse</td>
		</tr>
		<tr height="40">
			<td dir="LTR" height="40" style="height:40px;width:163px;">Agarose</td>
			<td style="width:132px;">Euromex</td>
			<td style="width:155px;">LM3 (1670-B, Tg 25.7C, Tm 64C)</td>
			<td style="width:149px;"></td>
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		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:163px;">Sucrose</td>
			<td style="width:132px;">Sigma</td>
			<td>84097-1kg</td>
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		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:163px;">Glucose&#160;</td>
			<td style="width:132px;">Sigma</td>
			<td>G8270-1kg</td>
			<td style="width:149px;"></td>
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			<td dir="LTR" height="20" style="height:20px;width:163px;">Trehalose</td>
			<td style="width:132px;">Sigma</td>
			<td>T9531-25G</td>
			<td style="width:149px;"></td>
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		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:163px;">KCl</td>
			<td style="width:132px;">Sigma</td>
			<td>P9333-1kg</td>
			<td style="width:149px;"></td>
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		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:163px;">Hepes</td>
			<td style="width:132px;">Sigma</td>
			<td>H3375-100G</td>
			<td style="width:149px;"></td>
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		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:163px;">TRIS</td>
			<td style="width:132px;">Euromex</td>
			<td>EU0011-A</td>
			<td style="width:149px;"></td>
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		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:163px;">Osmometer</td>
			<td style="width:132px;">Wescor</td>
			<td>Vapro</td>
			<td style="width:149px;"></td>
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		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:163px;">pH meter</td>
			<td style="width:132px;">Schott instruments</td>
			<td>Lab850</td>
			<td style="width:149px;"></td>
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		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:163px;">Sonicator</td>
			<td style="width:132px;">Elmasonic</td>
			<td>S 180 H</td>
			<td style="width:149px;"></td>
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		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:163px;">Syringe filter 0.2&#181;m</td>
			<td style="width:132px;">Sartorius steim</td>
			<td>Minisart 16532</td>
			<td style="width:149px;"></td>
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		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:163px;">Function generator</td>
			<td style="width:132px;">TTi</td>
			<td>TG315</td>
			<td style="width:149px;"></td>
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		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:163px;">Platinum wires</td>
			<td style="width:132px;">Goodfellow</td>
			<td>LS413074</td>
			<td style="width:149px;">99.99+%, d=0.5mm</td>
		</tr>
		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:163px;">Polytetrafluoroethylene</td>
			<td style="width:132px;">Goodfellow</td>
			<td></td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="40">
			<td dir="LTR" height="40" style="height:40px;width:163px;">Dow Corning &#39;high vacuum grease&#39;</td>
			<td style="width:132px;">VWR</td>
			<td>1597418</td>
			<td style="width:149px;"></td>
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		<tr height="40">
			<td dir="LTR" height="40" style="height:40px;width:163px;">sealing paste</td>
			<td style="width:132px;">Vitrex medical A/S, Denmark</td>
			<td>REF 140014</td>
			<td style="width:149px;">Sigillum Wax</td>
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			<td dir="LTR" height="40" style="height:40px;width:163px;">Cover slides 22x40 mm No1,5</td>
			<td style="width:132px;">VWR&#160;</td>
			<td>631-0136</td>
			<td style="width:149px;"></td>
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		<tr height="40">
			<td dir="LTR" height="40" style="height:40px;width:163px;">Cover slides 22x22 mm No1,5</td>
			<td style="width:132px;">VWR&#160;</td>
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			<td style="width:149px;"></td>
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			<td style="width:132px;">Harrick</td>
			<td>PDC-32G</td>
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			<td style="width:132px;">Falcon BD</td>
			<td>REF 353001</td>
			<td style="width:149px;">3.5 cmx1cm</td>
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			<td dir="LTR" height="20" style="height:20px;width:163px;">patch clamp amplifier</td>
			<td style="width:132px;">Axon instruments</td>
			<td>Multiclamp700B</td>
			<td style="width:149px;"></td>
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		<tr height="40">
			<td dir="LTR" height="40" style="height:40px;width:163px;">DAQ Card</td>
			<td style="width:132px;">National Instruments</td>
			<td>PCI-6221</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="40">
			<td dir="LTR" height="40" style="height:40px;width:163px;">Labview</td>
			<td style="width:132px;">National Instruments</td>
			<td>version 8.6</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="60">
			<td dir="LTR" height="60" style="height:60px;width:163px;">Glass pipettes boro silicate OD 1mm ID 0.58mm</td>
			<td style="width:132px;">Harvard Apparatus</td>
			<td>GC100-15</td>
			<td style="width:149px;"></td>
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		<tr height="40">
			<td dir="LTR" height="40" style="height:40px;width:163px;">Electrode holder</td>
			<td style="width:132px;">Warner Instruments&#160;</td>
			<td>Q45W-T10P</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:163px;">Micromanipulator</td>
			<td style="width:132px;">Sutter&#160;</td>
			<td>MP-285</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:163px;">pipette puller</td>
			<td style="width:132px;">Sutter&#160;</td>
			<td>P-2000&#160;</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:163px;">Camera</td>
			<td style="width:132px;">ProSilica&#160;</td>
			<td>GC1380</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:163px;">Zeiss microscope</td>
			<td style="width:132px;">Zeiss</td>
			<td>Axiovert 135</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="60">
			<td dir="LTR" height="60" style="height:60px;width:163px;">Objective</td>
			<td style="width:132px;">Zeiss</td>
			<td></td>
			<td style="width:149px;">40x long working distance, Phase contrast</td>
		</tr>
		<tr height="40">
			<td dir="LTR" height="40" style="height:40px;width:163px;">Objective&#160;</td>
			<td style="width:132px;">Zeiss</td>
			<td></td>
			<td style="width:149px;">100x Plan-Apochromat NA 1.3</td>
		</tr>
		<tr height="40">
			<td dir="LTR" height="40" style="height:40px;width:163px;">Filterset GFP (for Alexa-488)</td>
			<td style="width:132px;">Horiba</td>
			<td>XF100-3</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="40">
			<td dir="LTR" height="40" style="height:40px;width:163px;">Filterset Cy3 (for TexasRed)</td>
			<td style="width:132px;">Horiba</td>
			<td>XF101-2</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:163px;">beta-casein</td>
			<td style="width:132px;">Sigma&#160;</td>
			<td>C6905-1G</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="40">
			<td dir="LTR" height="40" style="height:40px;width:163px;">confocal microscope</td>
			<td style="width:132px;">Nikon Eclipse TE 2000-E</td>
			<td></td>
			<td style="width:149px;">D-Eclipse C1 confocal head</td>
		</tr>
		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:163px;">Objective</td>
			<td style="width:132px;">Nikon</td>
			<td></td>
			<td style="width:149px;">Plan Fluor 100&#215; NA1.3</td>
		</tr>
		<tr height="60">
			<td dir="LTR" height="60" style="height:60px;width:163px;">matlab</td>
			<td style="width:132px;">Mathworks</td>
			<td></td>
			<td style="width:149px;">for image processing and analysis of the current traces</td>
		</tr>
	</tbody>
</table>

reconstitution of a transmembrane protein, the voltage-gated ion channel, kvap, into giant unilamellar vesicles for microscopy and patch clamp studies

Giant Unilamellar Vesicles (GUVs) are a popular biomimetic system for studying membrane associated phenomena. However, commonly used protocols to grow GUVs must be modified in order to form GUVs containing functional transmembrane proteins. This article describes two dehydration-rehydration methods &#8212; electroformation and gel-assisted swelling &#8212; to form GUVs containing the voltage-gated potassium channel, KvAP. In both methods, a solution of protein-containing small unilamellar vesicles is partially dehydrated to form a stack of membranes, which is then allowed to swell in a rehydration buffer. For the electroformation method, the film is deposited on platinum electrodes so that an AC field can be applied during film rehydration. In contrast, the gel-assisted swelling method uses an agarose gel substrate to enhance film rehydration. Both methods can produce GUVs in low (e.g., 5 mM) and physiological (e.g., 100 mM) salt concentrations. The resulting GUVs are characterized via fluorescence microscopy, and the function of reconstituted channels measured using the inside-out patch-clamp configuration. While swelling in the presence of an alternating electric field (electroformation) gives a high yield of defect-free GUVs, the gel-assisted swelling method produces a more homogeneous protein distribution and requires no special equipment.

The growth of GUVs can be quickly evaluated by examining the growth chamber under the microscope. For electroformation, the GUVs tend to grow in bunches along the platinum wires, as shown in Figure 4. During gel-assisted swelling, GUVs appear as spherical structures that rapidly grow and fuse together (Figure 5).
Defect-free GUVs are more easily identified and evaluated after transferring to an observation chamber. Calibration measurements are needed to rigoro...

Watch this Scientific Journal Video about Reconstitution of a Transmembrane Protein, the Voltage-gated Ion Channel, KvAP, into Giant Unilamellar Vesicles for Microscopy and Patch Clamp Studies at JoVE

Reconstitution of a Transmembrane Protein, the Voltage-gated Ion Channel, KvAP, into Giant Unilamellar Vesicles for Microscopy and Patch Clamp Studies

The reconstitution of the transmembrane protein, KvAP, into giant unilamellar vesicles (GUVs) is demonstrated for two dehydration-rehydration methods &#8212; electroformation, and gel-assisted swelling. In both methods, small unilamellar vesicles containing the protein are fused together to form GUVs that can then be studied by fluorescence microscopy and patch-clamp electrophysiology.

Giant Unilamellar Vesicles (GUVs) are a popular biomimetic system for studying membrane associated phenomena. However, commonly used protocols to grow ...

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