Procedures for complete reconstitution of a prototype voltage-gated potassium channel into lipid membranes are described. The reconstituted channels are suitable for biochemical assays, electrical recordings, ligand screening and electron crystallographic studies. These methods may have general applications to the structural and functional studies of other membrane proteins.
To study the lipid-protein interaction in a reductionistic fashion, it is necessary to incorporate the membrane proteins into membranes of well-defined lipid composition. We are studying the lipid-dependent gating effects in a prototype voltage-gated potassium (Kv) channel, and have worked out detailed procedures to reconstitute the channels into different membrane systems. Our reconstitution procedures take consideration of both detergent-induced fusion of vesicles and the fusion of protein/detergent micelles with the lipid/detergent mixed micelles as well as the importance of reaching an equilibrium distribution of lipids among the protein/detergent/lipid and the detergent/lipid mixed micelles. Our data suggested that the insertion of the channels in the lipid vesicles is relatively random in orientations, and the reconstitution efficiency is so high that no detectable protein aggregates were seen in fractionation experiments. We have utilized the reconstituted channels to determine the conformational states of the channels in different lipids, record electrical activities of a small number of channels incorporated in planar lipid bilayers, screen for conformation-specific ligands from a phage-displayed peptide library, and support the growth of 2D crystals of the channels in membranes. The reconstitution procedures described here may be adapted for studying other membrane proteins in lipid bilayers, especially for the investigation of the lipid effects on the eukaryotic voltage-gated ion channels.
Cells exchange materials and information with their environment through the functions of specific membrane proteins 1. Membrane proteins in cell membranes function as pumps, channels, receptors, intramembrane enzymes, linkers and structural supporters across membranes. Mutations that affect the membrane proteins have been related to many human diseases. In fact, many membrane proteins have been the primary drug targets because they are important and easily accessible in cell membranes. It is therefore very important to understand the structure and function of various membrane proteins in membranes, and make it possible to devise novel methods to alleviate the detrimental effects from the mutant proteins in human diseases.
Lipids surround all membrane proteins integrated in bilayers 2, 3. In eukaryotic membranes, the various different types of lipids are known to be organized into microdomains 4, 5. Many membrane proteins were shown to be distributed among these microdomains as well as the bulky fluid phase of membranes 3, 6. The mechanism underlying the organization of the microdomains and the delivery of membrane proteins into them and the physiological significance of such distributions are clearly important but remain poorly understood. One major technical difficulty in studying the lipid effects on membrane proteins is the reliable reconstitution of biochemically purified membrane proteins into membranes of well-controlled lipid composition so that almost all reconstituted proteins are functional 7. In the past few years, we developed methods to reconstitute the prototype voltage-gated potassium channel from A. pernix (KvAP) into various membrane systems for structural and functional studies 8-10. The data from others and us together showed that the lipids are likely a determinant in the conformational changes of the voltage-sensing domains of a voltage-gated ion channel and may shape the structures of some of these channels 11. In the next, we will provide a detailed description of our methods and will offer critical technical tips that will likely ensure the successful reproduction of our results as well as the extension of our methods to the studies of other membrane proteins.
1. Expression and Purification of KvAP Channel (Figure 1)
2. Ion Channel Reconstitution
2.1. Liposome preparation and detergent-induced fusion of vesicles
Prior to the lipid preparation, wash a 14 ml disposable glass test-tube, a screw-capped glass tube, and a 250 μl glass syringe with chloroform. Pour out ~10 ml chloroform into the testtube.
2.2. Removal of the detergents to form proteoliposomes
2.3. Storage of the proteoliposome and the quality control
3. Applications of the Reconstituted Channel-containing Vesicles
3.1. Functional study of the ion channel activities in black lipid membranes.
Preparation of needed materials.
Electrical recordings from KvAP channels in lipid bilayers
3.2. Screening for conformation-specific ligands against channels in vesicles
3.3. Crystallization of KvAP channels in membranes for structure determination 27-29
The general flow of the experiments for purifying the KvAP channel into biochemical homogeneity is described in Figure 1A. Typical samples during the expression and purification of the protein is showed in the SDS-PAGE gel in Figure 1B. The protein after the IMAC purification is relatively pure. The yield of the KvAP channel is about 1.0 mg/Liter culture.
Solubilization of lipid vesicles with detergents needs to be worked out for each pair of lipid vs. detergent. The solubilization of small unilamellar vesicles of POPE/POPE by DM is presented in Figure 2A, the results from the vesicles floatation are usually fairly straightforward. Typical results in the SDS-PAGE assay of the fractions from the gradients are showed in Figure 2B. In the sucrose density gradient, the channel-containing POPE/POPG vesicles usually are concentrated at the interface between the 10% layer and the 35% layer. The KvAP-containing DOTAP or DOGS vesicles are lighter and usually at the interface tween 5% and 10% (slightly penetrated into the 10% layer). If there are a significant fraction of multilamellar vesicles, they usually are whitish and concentrated below the 10-35% interface. We found that this usually happens when the protein-detergent-lipid mixture was not incubated long enough to reach good distribution of the lipids.
Electrical recordings from KvAP channels incorporated in planar lipid bilayers are shown in Figure 3. The typical current trace shows that at -80 mV, the channels are quiet. Switching to +80 mV leads to a quick capacitance peak (the sharp one at the time of voltage switching). A slow rising phase of the current suggests that the channels become active and are able to conduct outward potassium current. After the peak of the rising phase, the current starts to decrease, a step named inactivation. The inactivation takes a few hundred milliseconds to complete. Once the voltage is switched back to -80 mV, there is a returning phase after the downward capacitance peak, which reflects the closing of the open channels and is called the deactivation (Figure 3C).
In the middle of the phage screen, we tested the activity of the amplified phages on the KvAP channels in the bilayers of POPE/POPG. After 12 selection cycles, the amplified phages inhibited the channel activities (Figure 4B, selected phage). But the starting phage-displayed library did not show any effect on the channels (Figure 4B, control phage). Even though the activities of the selected phages were not very high because only a low concentration of the positive phages were around, the clear, reproducible effects suggested that there are active, positive clones that exhibit high-binding affinity, should be selectively enriched during the remaining screening cycles and once enriched, are likely to have strong inhibitory activities on channels in membranes.
In the early stage of screening the 2D crystals, we saw small lattices in many small vesicles. With optimization, some large sheets showed up with a lot of small vesicles around (Figure 5A). The cryoEM images of these crystals always showed a lot of local defects (Figure 5B), suggesting that further optimization is required to obtain better crystals. Once the crystals were well optimized, they exhibited sharp edges, and appeared as single sheets. At high magnification, the individual units are clearly much better ordered (Figure 5C).
Name of Reagent/Material | Company | Catalog Number | Comments |
Tryptone | RPI Corp. | T60060 | |
Yeast Extract | RPI Corp. | Y20020 | |
NaCl | Fisher | S271-3 | |
Tris Base | RPI Corp. | T60040 | |
Potassium Chloride | Fisher | BP366-500 | |
n-Dodecyl-β-D-Maltoside | Affymetrix | D322S | Sol-grade |
n-Octyl-β-D-Glucoside | Affymetrix | O311 | Ana-grade |
Aprotinin | RPI Corp. | A20550-0.05 | |
Leupeptin | RPI Corp. | L22035-0.025 | |
Pepstatin A | RPI Corp. | P30100-0.025 | |
PMSF | SIGMA | P7626 | |
Dnase I | Roche | 13407000 | |
Bio-Bead SM-2 | Bio-Rad | 152-3920 | |
HEPES | RPI Corp. | H75030 | |
POPE | Avanti Polar Lipids | 850757C | |
POPG | Avanti Polar Lipids | 840457C | |
DOGS | Avanti Polar Lipids | 870314C | |
DMPC | Avanti Polar Lipids | 850345C | |
Biotin-DOPE | Avanti Polar Lipids | 870282C | |
DOTAP | Avanti Polar Lipids | 890890C | |
NeutrAvidin agarose beads | Piercenet | 29200 | |
Dialysis Tubing | Spectrum Laboratories, Inc | 132-570 | |
Pentane | Fisher | R399-1 | |
Decane | TCI America | D0011 | |
MTS-PEG5000 | Toronto Research Chemicals | M266501 |
Table 1. List of reagents and materials.
|
Table 2. Equipment needed.
Buffer name | Contents |
IMAC Lysis buffer | 50 mM Tris (pH 8.0), 100 mM KCl |
IMAC Wash buffer | 50 mM Tris (pH 8.0), 100 mM KCl, and 5.0 mM DM |
IMAC Elution buffer | 50 mM Tris (pH 8.0), 100 mM KCl, 5.0 mM DM, and 300 mM imidazole |
SDS-sample buffer (5X) (nonreducing) | 60 mM Tris-HCl (pH 6.8), 25% glycerol, 2.0% SDS, 0.10% bromophenol blue. (1.0% 2-mercaptoethanol added to make the reducing buffer). |
Stacking gel buffer for SDS-PAGE(4X) | 0.50 M Tris-HCl (pH 6.8), 0.40% SDS |
Resolution gel buffer for SDS-PAGE (4X) | 1.5 M Tris-HCl (pH 8.8), 0.40% SDS |
FPLC equilibration | 20 mM Tris pH 8.0, 100 mM KCl, and 5.0 mM DM |
Liposome dialysis | 10 mM HEPES, 100 mM KCl |
Table 3. Buffer name and contents.
Figure 1. Preparation of KvAP for reconstitution. A. General work flow of protein expression, purification and reconstitution. B. Biochemical purification of the protein. Induced culture of E. coli XL1-blue expressing KvAP was processed and KvAP purified. Twenty microliters of cell cultures before (lane 1) or after (lane 2) induction, the detergent extract (lane 3), flow-through from IMAC chromatography (lane 4), two washing steps (lanes 5.6), and 5.0 μg of total protein after the 300 mM imidazole elution (lane 7) and 5.0 μg of the KvAP tetramer out of the size exclusion FPLC (lane 8) were subjected to non-reducing 12% SDS-PAGE and Coomassie blue staining.
Figure 2. Reconstitution of KvAP in POPE/POPG vesicles. A. Vesicle fusion and solubilization as a function of detergent concentrations. Absorbance at 410 nm was used to monitor the light scattering of vesicles (baseline subtracted to no detergent fraction). Lipids (POPE/POPG 3:1) were 10 mg/ml. The small unilamellar vesicles after strong sonication, is monodispersed and has weak scattering due to their sizes of 30-50 nm in diameter. When the detergents were introduced, they distributed between solution phase and the lipid membrane phase. Due to the strong curvature in the small unilamellar vesicles, the introduced detergents trigger vesicle fusion and the release of the curvature (thus the low surface potential energy). The fused vesicles are larger in size and show stronger scattering at 410 nm. The rising phase of the peak (gray-colored area) therefore reflects the detergent-induced fusion, a good regime for protein micelle fusion to the vesicles. The concentration of the detergent (DM as an example here) right at the absorption peak was indeed chosen for our reconstitution process. B. Fifty microliters of reconstituted KvAP vesicles (KvAP 0.5 mg/ml) was separated by sucrose density gradient. Samples of 100 μl fractions from top to bottom (1~9) of sucrose gradient were assayed in a 12% reducing SDS-PAGE. The gel was Coomassie blue stained. Lane 3 contained ~2 μg KvAP.
Figure 3. Electrical activity of KvAP channel in reconstituted bilayers. A. Recording set-up inside a Faraday cage, 1; two electrodes, 2; trans side, 3; cis side, 4; salt bridge. B. Magnified section of the thinned portion at one side of the bilayer cup showing a hole 5, which is used for making membranes. C. Electrical activity of KvAP channels that were fused into the black lipid membrane was recorded. While being held at -80 mV, the membrane potential was pulsed to 80 mV for 150 msec, and then changed back to the holding potential. The ionic current was recorded in the voltage-clamp mode using an Axopatch 200B amplifier.
Figure 4. Screening for conformation-specific ligands from a phage-displayed library. A. Scheme behind the screening against ion channels reconstituted in vesicles. The vesicles are doped with biotin-DOPE, and they can be pulled out of solution by NeutrAvidin beads. The conformation of the KvAP channel is controlled by specific lipids. Negative selections against beads, empty vesicles and vesicles with channels in a different conformation are done before the phages are incubated with the channels in the target conformational state (in DOTAP or DOGS as an example here). The selected phages can be amplified and tested against channels in bilayers. B. Testing of the selected phages in the middle of the screen. Electrical activity of KvAP in the bilayer at different time points after the addition of the selected phages: black trace – before the addition; yellow trace – 2 min after; red trace – 10 min after the addition. Top: The starting phage library (total ~1010 phages added to the bilayer) was tested on channels in bilayer, and was found to have no detectable activity because each phage clone has only about 100 copies. Bottom: After 12 selection cycles, the phages were still a mixture of different clones. Adding about 1010 phages to the channels in the bilayer lead to the clear inhibition of channel activity, suggesting that there are positive clones that should have high affinity. Click here to view larger figure.
Figure 5. Two-dimensional crystallization of KvAP in membranes. A. Image of negatively stained single layer 2D crystals in the middle of crystal optimization. The crystals were stained with 6.0% ammonium molybdate, pH 6.4 plus 0.50% trehalose. Due to the sugar, they sometimes piled up with each other. The black square box designates an area that gives rise to the diffraction pattern shown on the right side with diffraction spots going to ~20 Â. B. CryoEM image of a 2D crystal from a sample similar to the one used in (A). The specimen was mixed with 3% trehalose and frozen by direct plunging, and imaged under a cryoEM. The image was obtained at 50,000 x. It was clear that there were local defects in the crystal packing. C. CryoEM image of a further-optimized crystal. The specimen was embedded in 0.75% tannin and 10% trehalose and the image was taken at 50,000 X. The straight lines and the tight packing suggested that the channels were well ordered in this type of crystals. The two black arrows mark the square lattice.
Boxes:
Box 1. Preparation of IMAC column
Box 2. Purification of Fv
Fv Transformation – Day 1
Expression of Fv – Day 2
Releasing the Fv molecules from the bacteria – Day 3
IMAC purification and FPLC – Day 4
The reconstitution of the KvAP channels into different membranes has been used in several studies 8-10. Following the idea of ensuring the distribution of lipids between detergent/lipid mixed micelles and the protein/detergent/lipid mixed micelles, we are able to reach nearly complete reconstitution of the KvAP into membranes made of very different lipids. Each tetrameric KvAP channel needs ~100 lipid molecules to fully cover its transmembrane domain. The essential requirement is to allow enough lipid molecules to fuse into the protein/lipid/detergent micelles before the detergents are removed. Our standard conditions (0.5 mg protein and 5.0 mg lipids) ensure that there are on average ~1,000 lipid molecules per protein molecule. Our floatation experiment and biochemical experiments confirmed that the reconstitution is almost complete. Electrical recordings from channels inserted into black lipid membranes, the screening of the reconstituted channels against a phage-displayed peptide library, and the growth of 2D crystals of the channels in membranes all demonstrate the successful applications of membrane reconstitution for various purposes.
The lipid-dependent conformational changes of the KvAP channels and the screening against a phage-displayed peptide library showcase a new avenue to screen for channel blockers or channel openers by biochemical methods instead of relying on electrophysiology to keep the channels in specific conformations 8. The success in our screen for conformation-specific binders suggests that the same strategy can be applied to find specific binders for the activated conformation. It is foreseeable that the reconstituted channels in vesicles can be used against single chain Fv libraries, Fab libraries, etc. Likewise, other membrane proteins can run through these operations and secure their tight binders that may be useful for various purposes. We believe that this new method will see more general applications in the future.
Reconstituted membrane systems will allow the elucidation of the chemical details behind the lipid effects on membrane proteins 11. Lipid-protein interaction has been known to be important for many membrane proteins, and has been subjected to multiple studies in the past 3. In the cell-based studies, manipulations can be implemented to change the specific components in the membranes and then the functional changes in the membrane proteins are associated with the structural and compositional changes in the membranes. Such connections are indirect and might result from multiple factors in the cell membranes that are not well characterized. In a reconstituted homogeneous membrane, it is more definitive in making connections between the structural and functional changes of the membrane proteins and the changes in lipid composition and membrane properties. Ultimately, to understand the chemical principles behind the lipid-protein interaction, we need to delineate the distribution of lipids around the transmembrane domain, and to understand the dynamic changes of these lipids right next to the proteins. A reconstituted system appears to be a reliable way toward such an understanding.
Reconstitution of membrane proteins requires the controlled removal of detergents from the protein/lipid/detergent mixed micelles, and the fusion of the mixed micelles into large ones that eventually turn into vesicles 30. Three different methods are being used for removing detergents, dialysis, beads, and cyclodextrin 14, 31, 32. But it remains difficult to achieve a well-controlled, gradual removal of detergents from a small volume 33, 34. An ideal method for detergent removal would take the detergents out of the aqueous phase evenly across the whole volume in a controllable pace, and should not exert strong interference on the reconstitution of bilayer membranes. Such a method might be able to change the speed and efficacy of reconstitution, and will likely enable the reconstitution in a small volume. A combination of slow-dilution and any of three conventional methods for detergent removal may approach this goal. Slow-dilution by introducing small amount of water into the protein/detergent/lipid mixture is a controllable way to evenly decrease the detergent concentration down to its CMC. The detergent removal afterwards is less critical for the vesicle formation, although still important for the fusion of small vesicles into large ones. Other ways to achieve controlled detergent removal still need to be conceived and developed.
Our reconstitution procedure takes consideration of lipid distribution among mixed micelles and the detergent-induced fusion of vesicles as well as the mixed micelles. Its success paves the way leading to a broader spectrum of applications of the reconstituted vesicles, much more than the three directions we presented. The adaptation of our procedure to other membrane proteins should not encounter major technical limit. Even though many membrane proteins have been reconstituted one way or the other, it has been difficult to achieve near complete reconstitution and to evaluate the functionality of the proteins from multiple different perspectives. Our efforts in the KvAP reconstitution suggest that our methods may allow full reconstitution and will be suitable for these purposes.
The authors have nothing to disclose.
The studies on KvAP in the Jiang lab have obtained significant help from Dr. Roderick MacKinnon’s laboratory at the Rockefeller University. Special thanks go to Dr. Kathlynn Brown and Michael McQuire for their advice and help on our phage-screen experiments. This work was supported by grants from NIH (GM088745 and GM093271 to Q-XJ) and AHA (12IRG9400019 to Q-XJ).
Name of Reagent/Material | Company | Catalog Number | Comments |
Tryptone | RPI Corp. | T60060 | |
Yeast Extract | RPI Corp. | Y20020 | |
NaCl | Fisher | S271-3 | |
Tris Base | RPI Corp. | T60040 | |
Potassium Chloride | Fisher | BP366-500 | |
n-Dodecyl-β-D-Maltoside | Affymetrix | D322S | Sol-grade |
n-Octyl-β-D-Glucoside | Affymetrix | O311 | Ana-grade |
Aprotinin | RPI Corp. | A20550-0.05 | |
Leupeptin | RPI Corp. | L22035-0.025 | |
Pepstatin A | RPI Corp. | P30100-0.025 | |
PMSF | SIGMA | P7626 | |
Dnase I | Roche | 13407000 | |
Bio-Bead SM-2 | Bio-Rad | 152-3920 | |
HEPES | RPI Corp. | H75030 | |
POPE | Avanti Polar Lipids | 850757C | |
POPG | Avanti Polar Lipids | 840457C | |
DOGS | Avanti Polar Lipids | 870314C | |
DMPC | Avanti Polar Lipids | 850345C | |
Biotin-DOPE | Avanti Polar Lipids | 870282C | |
DOTAP | Avanti Polar Lipids | 890890C | |
NeutrAvidin agarose beads | Piercenet | 29200 | |
Dialysis Tubing | Spectrum Laboratories, Inc | 132-570 | |
Pentane | Fisher | R399-1 | |
Decane | TCI America | D0011 | |
MTS-PEG5000 | Toronto Research Cemicals | M266501 |