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. 1982 Dec;79(24):7749–7753. doi: 10.1073/pnas.79.24.7749

Isolated-patch recording from liposomes containing functionally reconstituted chloride channels from Torpedo electroplax.

D W Tank, C Miller, W W Webb
PMCID: PMC347425  PMID: 6296849

Abstract

Small unilamellar vesicles formed from purified phospholids by detergent/dialysis methods may be enlarged to 30-microns diameter by freezing and thawing. Very-high-resistance seals were formed by applying a glass micropipette to the surface of these large liposomes, and single bilayer "patches" of membrane were isolated from the liposome surface while remaining sealed to the micropipette. The exogenous channel-forming peptides gramicidin and alamethicin induced characteristic single-channel fluctuation behavior in these excised patches held under voltage-clamp conditions. Large liposomes were formed from the small unilamellar vesicles made from cholate extracts of Torpedo electroplax plasma membrane vesicles. Isolated patches formed from these reconstituted membranes displayed current fluctuations due to single voltage-gated Cl- channels from non-innervated-face membranes; the properties of these Cl- channels are identical to those observed in planar bilayer membranes after direct insertion from native membranes. This liposome-patch method combines the advantages of membrane protein incorporation into liposomes with high-resolution electrical recording methods and may provide a generally applicable approach to the study of integral membrane channel proteins after solubilization and reconstitution.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Eisenberg M., Hall J. E., Mead C. A. The nature of the voltage-dependent conductance induced by alamethicin in black lipid membranes. J Membr Biol. 1973 Dec 31;14(2):143–176. doi: 10.1007/BF01868075. [DOI] [PubMed] [Google Scholar]
  2. Epstein M., Racker E. Reconstitution of carbamylcholine-dependent sodium ion flux and desensitization of the acetylcholine receptor from Torpedo californica. J Biol Chem. 1978 Oct 10;253(19):6660–6662. [PubMed] [Google Scholar]
  3. Fröhlich O. Asymmetry of the gramicidin channel in bilayers of asymmetric lipid composition: II. Voltage dependence of dimerization. J Membr Biol. 1979 Aug;48(4):385–401. doi: 10.1007/BF01869448. [DOI] [PubMed] [Google Scholar]
  4. Gordon L. G., Haydon D. A. Kinetics and stability of alamethicin conducting channels in lipid bilayers. Biochim Biophys Acta. 1976 Jul 1;436(3):541–556. doi: 10.1016/0005-2736(76)90439-9. [DOI] [PubMed] [Google Scholar]
  5. Hamill O. P., Marty A., Neher E., Sakmann B., Sigworth F. J. Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Arch. 1981 Aug;391(2):85–100. doi: 10.1007/BF00656997. [DOI] [PubMed] [Google Scholar]
  6. Hanke W., Eibl H., Boheim G. A new method for membrane reconstitution: fusion of protein-containing vesicles with planar bilayer membranes below lipid phase transition temperature. Biophys Struct Mech. 1981;7(3):131–137. doi: 10.1007/BF00539175. [DOI] [PubMed] [Google Scholar]
  7. Hladky S. B., Haydon D. A. Ion transfer across lipid membranes in the presence of gramicidin A. I. Studies of the unit conductance channel. Biochim Biophys Acta. 1972 Aug 9;274(2):294–312. doi: 10.1016/0005-2736(72)90178-2. [DOI] [PubMed] [Google Scholar]
  8. Horn R., Patlak J. Single channel currents from excised patches of muscle membrane. Proc Natl Acad Sci U S A. 1980 Nov;77(11):6930–6934. doi: 10.1073/pnas.77.11.6930. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Huganir R. L., Schell M. A., Racker E. Reconstitution of the purified acetylcholine receptor from Torpedo californica. FEBS Lett. 1979 Dec 1;108(1):155–160. doi: 10.1016/0014-5793(79)81199-0. [DOI] [PubMed] [Google Scholar]
  10. Kasahara M., Hinkle P. C. Reconstitution and purification of the D-glucose transporter from human erythrocytes. J Biol Chem. 1977 Oct 25;252(20):7384–7390. [PubMed] [Google Scholar]
  11. Kolb H. A., Bamberg E. Influence of membrane thickness and ion concentration on the properties of the gramicidin a channel. Autocorrelation, spectral power density, relaxation and single-channel studies. Biochim Biophys Acta. 1977 Jan 4;464(1):127–141. doi: 10.1016/0005-2736(77)90376-5. [DOI] [PubMed] [Google Scholar]
  12. Korenbrot J. I., Hwang S. B. Proton transport by bacteriorhodopsin in planar membranes assembled from air-water interface films. J Gen Physiol. 1980 Dec;76(6):649–682. doi: 10.1085/jgp.76.6.649. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Latorre R., Alvarez O. Voltage-dependent channels in planar lipid bilayer membranes. Physiol Rev. 1981 Jan;61(1):77–150. doi: 10.1152/physrev.1981.61.1.77. [DOI] [PubMed] [Google Scholar]
  14. Lindstrom J., Anholt R., Einarson B., Engel A., Osame M., Montal M. Purification of acetylcholine receptors, reconstitution into lipid vesicles, and study of agonist-induced cation channel regulation. J Biol Chem. 1980 Sep 10;255(17):8340–8350. [PubMed] [Google Scholar]
  15. Miller C. Voltage-gated cation conductance channel from fragmented sarcoplasmic reticulum: steady-state electrical properties. J Membr Biol. 1978 Apr 20;40(1):1–23. doi: 10.1007/BF01909736. [DOI] [PubMed] [Google Scholar]
  16. Miller C., White M. M. A voltage-dependent chloride conductance channel from Torpedo electroplax membrane. Ann N Y Acad Sci. 1980;341:534–551. doi: 10.1111/j.1749-6632.1980.tb47197.x. [DOI] [PubMed] [Google Scholar]
  17. Pick U. Liposomes with a large trapping capacity prepared by freezing and thawing of sonicated phospholipid mixtures. Arch Biochem Biophys. 1981 Nov;212(1):186–194. doi: 10.1016/0003-9861(81)90358-1. [DOI] [PubMed] [Google Scholar]
  18. Sakmann B., Boheim G. Alamethicin-induced single channel conductance fluctuations in biological membranes. Nature. 1979 Nov 15;282(5736):336–339. doi: 10.1038/282336a0. [DOI] [PubMed] [Google Scholar]
  19. Schindler H. Formation of planar bilayers from artificial or native membrane vesicles. FEBS Lett. 1980 Dec 15;122(1):77–79. doi: 10.1016/0014-5793(80)80405-4. [DOI] [PubMed] [Google Scholar]
  20. Sobel A., Weber M., Changeux J. P. Large-scale purification of the acetylcholine-receptor protein in its membrane-bound and detergent-extracted forms from Torpedo marmorata electric organ. Eur J Biochem. 1977 Oct 17;80(1):215–224. doi: 10.1111/j.1432-1033.1977.tb11874.x. [DOI] [PubMed] [Google Scholar]
  21. Walker J. W., McNamee M. G., Pasquale E., Cash D. J., Hess G. P. Acetylcholine receptor inactivation in Torpedo californica electroplax membrane vesicles. Detection of two processes in the millisecond and second time regions. Biochem Biophys Res Commun. 1981 May 15;100(1):86–90. doi: 10.1016/s0006-291x(81)80066-6. [DOI] [PubMed] [Google Scholar]
  22. White M. M., Miller C. A voltage-gated anion channel from the electric organ of Torpedo californica. J Biol Chem. 1979 Oct 25;254(20):10161–10166. [PubMed] [Google Scholar]
  23. White M. M., Miller C. Chloride permeability of membrane vesicles isolated from Torpedo californica electroplax. Biophys J. 1981 Aug;35(2):455–462. doi: 10.1016/S0006-3495(81)84801-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Wu W. C., Moore H. P., Raftery M. A. Quantitation of cation transport by reconstituted membrane vesicles containing purified acetylcholine receptor. Proc Natl Acad Sci U S A. 1981 Feb;78(2):775–779. doi: 10.1073/pnas.78.2.775. [DOI] [PMC free article] [PubMed] [Google Scholar]

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