JoVE Logo
Faculty Resource Center

Sign In

Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Biology

Reconstitution of a Kv Channel into Lipid Membranes for Structural and Functional Studies

Published: July 13th, 2013

DOI:

10.3791/50436

1Department of Cell Biology, University of Texas Southwestern Medical Center at Dallas
* These authors contributed equally

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 t....

Log in or to access full content. Learn more about your institution’s access to JoVE content here

1. Expression and Purification of KvAP Channel (Figure 1)

  1. Preparation Work - Day 0
    1. Rinse the glass flasks for the bacterial culture with deionized water (diH2O) and MilliQ H2O (MQH2O) to remove trace of detergent from general dishwashing.
    2. Autoclave 1,000 ml LB medium in 2.8 L Erlenmeyer flasks (total two-liter culture as an example here). Low hardness of the water was found to be important for the successful culture of the transformed ba.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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. deterge.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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 molecule.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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).

....

Log in or to access full content. Learn more about your institution’s access to JoVE content here

Name Company Catalog Number Comments
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

  1. Alberts, B., et al. . Molecular Biology of the Cell. , (2007).
  2. Lee, A. G. How lipids and proteins interact in a membrane: a molecular approach. Mol. Biosyst. 1, 203 (2005).
  3. Lee, A. G. How lipids affect the activities of integral membrane proteins. Biochim. Biophys. Acta. 1666, 62 (2004).
  4. Anderson, R. G. The caveolae membrane system. Annu. Rev. Biochem. 67, 199 (1998).
  5. Simons, K., Vaz, W. L. Model systems, lipid rafts, and cell membranes. Annu. Rev. Biophys. Biomol. Struct. 33, 269 (2004).
  6. Edidin, M. The state of lipid rafts: from model membranes to cells. Annu. Rev. Biophys. Biomol. Struct. 32, 257 (2003).
  7. Kapoor, R., Kim, J. H., Ingolfson, H., Andersen, O. S. Preparation of Artificial Bilayers for Electrophysiology Experiments. J. Vis. Exp. (20), e1033 (2008).
  8. Zheng, H., Liu, W., Anderson, L. Y., Jiang, Q. X. Lipid-dependent gating of a voltage-gated potassium channel. Nat. Commun. 2, 250 (2011).
  9. Schmidt, D., Jiang, Q. X., MacKinnon, R. Phospholipids and the origin of cationic gating charges in voltage sensors. Nature. 444, 775 (2006).
  10. Ruta, V., Jiang, Y., Lee, A., Chen, J., MacKinnon, R. Functional analysis of an archaebacterial voltage-dependent K+ channel. Nature. 422, 180 (2003).
  11. Jiang, Q. X., Gonen, T. The influence of lipids on voltage-gated ion channels. Curr. Opin. Struct. Biol. , 3408884 (2012).
  12. Artimo, P., et al. ExPASy: SIB bioinformatics resource portal. Nucleic Acids Res. 40, W597 (2012).
  13. Cladera, J., Rigaud, J. L., Villaverde, J., Dunach, M. Liposome solubilization and membrane protein reconstitution using Chaps and Chapso. Eur. J. Biochem. 243, 798 (1997).
  14. Levy, D., Bluzat, A., Seigneuret, M., Rigaud, J. L. A systematic study of liposome and proteoliposome reconstitution involving Bio-Bead-mediated Triton X-100 removal. Biochim. Biophys. Acta. 1025, 179 (1990).
  15. Young, H. S., Rigaud, J. L., Lacapere, J. J., Reddy, L. G., Stokes, D. L. How to make tubular crystals by reconstitution of detergent-solubilized Ca2(+)-ATPase. Biophys. J. 72, 2545 (1997).
  16. Levy, D., Gulik, A., Bluzat, A., Rigaud, J. L. Reconstitution of the sarcoplasmic reticulum Ca(2+)-ATPase: mechanisms of membrane protein insertion into liposomes during reconstitution procedures involving the use of detergents. Biochim. Biophys. Acta. 1107, 283 (1992).
  17. Cohen, F. S., Zimmerberg, J., Finkelstein, A. Fusion of phospholipid vesicles with planar phospholipid bilayer membranes. II. Incorporation of a vesicular membrane marker into the planar membrane. The Journal of General Physiology. 75, 251 (1980).
  18. Fuks, B., Homble, F. Permeability and electrical properties of planar lipid membranes from thylakoid lipids. Biophysical Journal. 66, 1404 (1994).
  19. Hanke, W., Schlue, W. -. R., Sattelle, D. B. Planar Lipid Bilayers. Methods and Applications. Biological Techniques. , 133 (1993).
  20. Tien, H. T. . Bilayer lipid membranes (BLM). Theory and Practice. , 655-65 (1974).
  21. Pagano, R. E., Ruysschaert, J. M., Miller, I. R. The molecular composition of some lipid bilayer membranes in aqueous solution. The Journal of Membrane Biology. 10, 11 (1972).
  22. Henn, F. A., Thompson, T. E. Properties of lipid bilayer membranes separating two aqueous phases: composition studies. Journal of Molecular Biology. 31, 227 (1968).
  23. Tao, X., MacKinnon, R. Functional analysis of Kv1.2 and paddle chimera Kv channels in planar lipid bilayers. J. Mol. Biol. 382, 24 (2008).
  24. Cohen, F. S., Akabas, M. H., Zimmerberg, J., Finkelstein, A. Parameters affecting the fusion of unilamellar phospholipid vesicles with planar bilayer membranes. The Journal of Cell Biology. 98, 1054 (1984).
  25. Smith, G. P., Petrenko, V. A. Phage Display. Chem. Rev. 97, 391-39 (1997).
  26. McGuire, M. J., Li, S., Brown, K. C. Biopanning of phage displayed peptide libraries for the isolation of cell-specific ligands. Methods Mol. Biol. 504, 291 (2009).
  27. Rigaud, J. L. Membrane proteins: functional and structural studies using reconstituted proteoliposomes and 2-D crystals. Braz. J. Med. Biol. Res. 35, 753 (2002).
  28. Chami, M., et al. Use of octyl beta-thioglucopyranoside in two-dimensional crystallization of membrane proteins. J. Struct. Biol. 133, 64 (2001).
  29. Kuhlbrandt, W. Two-dimensional crystallization of membrane proteins. Q. Rev. Biophys. 25, 1 (1992).
  30. Rigaud, J. L., Levy, D. Reconstitution of membrane proteins into liposomes. Methods Enzymol. 372, 65 (2003).
  31. Walz, T., Grigorieff, N. Electron Crystallography of Two-Dimensional Crystals of Membrane Proteins. J. Struct. Biol. 121 (2), 142 (1998).
  32. Signorell, G. A., Kaufmann, T. C., Kukulski, W., Engel, A., Remigy, H. W. Controlled 2D crystallization of membrane proteins using methyl-beta-cyclodextrin. J. Struct. Biol. 157, 321 (2007).
  33. Vink, M., Derr, K., Love, J., Stokes, D. L., Ubarretxena-Belandia, I. A high-throughput strategy to screen 2D crystallization trials of membrane proteins. J. Struct. Biol. 160, 295 (2007).
  34. Iacovache, I., et al. The 2DX robot: a membrane protein 2D crystallization Swiss Army knife. J. Struct. Biol. 169, 370 (2010).

This article has been published

Video Coming Soon

JoVE Logo

Privacy

Terms of Use

Policies

Research

Education

ABOUT JoVE

Copyright © 2024 MyJoVE Corporation. All rights reserved