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

Summary

The reconstitution of the transmembrane protein, KvAP, into giant unilamellar vesicles (GUVs) is demonstrated for two dehydration-rehydration methods — 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.

Abstract

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 — electroformation and gel-assisted swelling — 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.

Introduction

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 ~ 1–100 µm) have proven to be a very useful biomimetic system^2–7 as they are well suited for microscopy studies and micromanipulation^8–10. While there are many different protocols to produce GUVs, most fall into two categories — emulsion based approaches^11,12 and tec....

Protocol

1. Solution Preparation

1. Prepare 5 ml of ‘SUV buffer’ 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 µm syringe filter and divide into 1 ml aliquots which can be stored at -20 °C.
   NOTE: Additional details for reagents and instruments are given in the materials list.
2. Prepare 40 ml of GUV ‘Growth Buffer’ that will fill the GUV interior during film rehydration. For a ‘low salt’ growth, combine 5 mM KCl, 1 mM HEPES (pH 7.4) or 1 mM TRIS (pH 7.5), and ~400 mM sucrose. For a ‘physiological salt’ growth, combine 100 mM KCl, 5 mM H....

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
DPhPC (1,2-diphytanoyl-sn-glycero-3-phosphocholine)	Avanti Polar Lipids	850356P
Egg PC L-α-phosphatidylcholine (Egg, Chicken)	Avanti Polar Lipids	840051P
Egg PA L-α-phosphatidic acid (Egg, Chicken)	Avanti Polar Lipids	840101P
18:1 (Δ9-cis) PC (DOPC) 1,2-dioleoyl-sn-glycero-3-phosphocholine	Avanti Polar Lipids	850375P
Cholesterol (ovine wool, >98%)	Avanti Polar Lipids	700000P
TRed-DHPE	Invirtogen	T-1395MP	labeled lipid
BPTR-Ceramide	Invirtogen	D-7540	labeled lipid
Choloroform	VWR	22711.290	AnalaR Normapur
Acetone	VWR	20066.296	AnalaR Normapur
Ethanol	VWR	20821.330	AnalaR Normapur
Kimwipe	Kimtech	7552
Hamilton syringes	Hamilton Bonaduz AG		diverse
Amber vials and Teflon cups	Sigma	SU860083 and SU860076
Parafilm	VWR	291-1214
Microcentrifuge tube	Eppendorf		diverse
Agarose	Euromex	LM3 (1670-B, Tg 25.7C, Tm 64C)
Sucrose	Sigma	84097-1kg
Glucose	Sigma	G8270-1kg
Trehalose	Sigma	T9531-25G
KCl	Sigma	P9333-1kg
Hepes	Sigma	H3375-100G
TRIS	Euromex	EU0011-A
Osmometer	Wescor	Vapro
pH meter	Schott instruments	Lab850
Sonicator	Elmasonic	S 180 H
Syringe filter 0.2 µm	Sartorius steim	Minisart 16532
Function generator	TTi	TG315
Platinum wires	Goodfellow	LS413074	99.99+%, d = 0.5 mm
Polytetrafluoroethylene	Goodfellow
Dow Corning 'high vacuum grease'	VWR	1597418
sealing paste	Vitrex medical A/S, Denmark	REF 140014	Sigillum Wax
Cover slides 22 x 40 mm No1.5	VWR	631-0136
Cover slides 22 x 22 mm No1.5	VWR	631-0125
Plasma cleaner	Harrick	PDC-32G	airplasma, setting 'high'
Petri dishes	Falcon BD	REF 353001	3.5 cm x 1 cm
patch clamp amplifier	Axon instruments	Multiclamp700B
DAQ Card	National Instruments	PCI-6221
Labview	National Instruments	version 8.6
Glass pipettes boro silicate OD 1 mm ID 0.58 mm	Harvard Apparatus	GC100-15
Electrode holder	Warner Instruments	Q45W-T10P
Micromanipulator	Sutter	MP-285
Pipette puller	Sutter	P-2000
Camera	ProSilica	GC1380
Zeiss microscope	Zeiss	Axiovert 135
Objective	Zeiss		40X long working distance, Phase contrast
Objective	Zeiss		100X Plan-Apochromat NA 1.3
Filterset GFP (for Alexa-488)	Horiba	XF100-3
Filterset Cy3 (for TexasRed)	Horiba	XF101-2
beta-casein	Sigma	C6905-1G
Confocal microscope	Nikon Eclipse TE 2000-E		D-Eclipse C1 confocal head
Objective	Nikon		Plan Fluor 100X NA 1.3
Matlab	Mathworks		for image processing and analysis of the current traces