Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1991 Oct 1;115(1):141–150. doi: 10.1083/jcb.115.1.141

Isolation and purification of gap junction channels

PMCID: PMC2289915  PMID: 1655801

Abstract

This paper reports methods we have developed to solubilize gap junction channels, or connexons, from isolated gap junctions and to purify them in milligram quantities. Two sources of material are used: rat liver gap junctions and gap junctions produced by infecting insect cells with a baculovirus containing the cDNA for human liver beta 1 protein (connexin 32). Complete solubilization is obtained with long chain detergents (lauryl dimethyl amineoxide, dodecyl maltoside) and requires high ionic strength and high pH as well as reducing conditions. The purification involves chromatography on hydroxylapatite and gel filtration on Superose 6. A homogeneous product is indicated by a single band on a silver-stained gel and a homogeneous population of doughnut-shaped particles under the electron microscope. These particles have hexameric symmetry. The purified connexons have a tendency to form aggregates: filaments and sheets. The filaments grow by end-to-end association of connexons and are nonpolar, suggesting that the connexons are paired as in the cell-to-cell channel. The sheets grow by lateral association of the filaments.

Full Text

The Full Text of this article is available as a PDF (1.7 MB).

Selected References

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

  1. Bennett M. V., Barrio L. C., Bargiello T. A., Spray D. C., Hertzberg E., Sáez J. C. Gap junctions: new tools, new answers, new questions. Neuron. 1991 Mar;6(3):305–320. doi: 10.1016/0896-6273(91)90241-q. [DOI] [PubMed] [Google Scholar]
  2. Beyer E. C., Paul D. L., Goodenough D. A. Connexin43: a protein from rat heart homologous to a gap junction protein from liver. J Cell Biol. 1987 Dec;105(6 Pt 1):2621–2629. doi: 10.1083/jcb.105.6.2621. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Cascio M., Gogol E., Wallace B. A. The secondary structure of gap junctions. Influence of isolation methods and proteolysis. J Biol Chem. 1990 Feb 5;265(4):2358–2364. [PubMed] [Google Scholar]
  4. Crowther R. A., Amos L. A. Harmonic analysis of electron microscope images with rotational symmetry. J Mol Biol. 1971 Aug 28;60(1):123–130. doi: 10.1016/0022-2836(71)90452-9. [DOI] [PubMed] [Google Scholar]
  5. Ebihara L., Beyer E. C., Swenson K. I., Paul D. L., Goodenough D. A. Cloning and expression of a Xenopus embryonic gap junction protein. Science. 1989 Mar 3;243(4895):1194–1195. doi: 10.1126/science.2466337. [DOI] [PubMed] [Google Scholar]
  6. Finbow M. E., Shuttleworth J., Hamilton A. E., Pitts J. D. Analysis of vertebrate gap junction protein. EMBO J. 1983;2(9):1479–1486. doi: 10.1002/j.1460-2075.1983.tb01611.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Gimlich R. L., Kumar N. M., Gilula N. B. Differential regulation of the levels of three gap junction mRNAs in Xenopus embryos. J Cell Biol. 1990 Mar;110(3):597–605. doi: 10.1083/jcb.110.3.597. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Gimlich R. L., Kumar N. M., Gilula N. B. Sequence and developmental expression of mRNA coding for a gap junction protein in Xenopus. J Cell Biol. 1988 Sep;107(3):1065–1073. doi: 10.1083/jcb.107.3.1065. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gruijters W. T., Kistler J., Bullivant S., Goodenough D. A. Immunolocalization of MP70 in lens fiber 16-17-nm intercellular junctions. J Cell Biol. 1987 Mar;104(3):565–572. doi: 10.1083/jcb.104.3.565. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Guthrie S. C., Gilula N. B. Gap junctional communication and development. Trends Neurosci. 1989 Jan;12(1):12–16. doi: 10.1016/0166-2236(89)90150-1. [DOI] [PubMed] [Google Scholar]
  11. Henderson D., Eibl H., Weber K. Structure and biochemistry of mouse hepatic gap junctions. J Mol Biol. 1979 Aug 5;132(2):193–218. doi: 10.1016/0022-2836(79)90391-7. [DOI] [PubMed] [Google Scholar]
  12. Hertzberg E. L. A detergent-independent procedure for the isolation of gap junctions from rat liver. J Biol Chem. 1984 Aug 10;259(15):9936–9943. [PubMed] [Google Scholar]
  13. Hertzberg E. L., Gilula N. B. Isolation and characterization of gap junctions from rat liver. J Biol Chem. 1979 Mar 25;254(6):2138–2147. [PubMed] [Google Scholar]
  14. Kistler J., Bullivant S. Dissociation of lens fibre gap junctions releases MP70. J Cell Sci. 1988 Nov;91(Pt 3):415–421. doi: 10.1242/jcs.91.3.415. [DOI] [PubMed] [Google Scholar]
  15. Kistler J., Kirkland B., Bullivant S. Identification of a 70,000-D protein in lens membrane junctional domains. J Cell Biol. 1985 Jul;101(1):28–35. doi: 10.1083/jcb.101.1.28. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kumar N. M., Gilula N. B. Cloning and characterization of human and rat liver cDNAs coding for a gap junction protein. J Cell Biol. 1986 Sep;103(3):767–776. doi: 10.1083/jcb.103.3.767. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  18. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  19. Makowski L., Caspar D. L., Phillips W. C., Goodenough D. A. Gap junction structures. II. Analysis of the x-ray diffraction data. J Cell Biol. 1977 Aug;74(2):629–645. doi: 10.1083/jcb.74.2.629. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Manjunath C. K., Page E. Rat heart gap junctions as disulfide-bonded connexon multimers: their depolymerization and solubilization in deoxycholate. J Membr Biol. 1986;90(1):43–57. doi: 10.1007/BF01869685. [DOI] [PubMed] [Google Scholar]
  21. Mazet F., Mazet J. L. Restoration of gap junction-like structure after detergent solubilization of the proteins from liver gap junctions. Exp Cell Res. 1990 Jun;188(2):312–315. doi: 10.1016/0014-4827(90)90175-a. [DOI] [PubMed] [Google Scholar]
  22. Milks L. C., Kumar N. M., Houghten R., Unwin N., Gilula N. B. Topology of the 32-kd liver gap junction protein determined by site-directed antibody localizations. EMBO J. 1988 Oct;7(10):2967–2975. doi: 10.1002/j.1460-2075.1988.tb03159.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Nicholson B., Dermietzel R., Teplow D., Traub O., Willecke K., Revel J. P. Two homologous protein components of hepatic gap junctions. Nature. 1987 Oct 22;329(6141):732–734. doi: 10.1038/329732a0. [DOI] [PubMed] [Google Scholar]
  24. Paul D. L. Molecular cloning of cDNA for rat liver gap junction protein. J Cell Biol. 1986 Jul;103(1):123–134. doi: 10.1083/jcb.103.1.123. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Revel J. P., Karnovsky M. J. Hexagonal array of subunits in intercellular junctions of the mouse heart and liver. J Cell Biol. 1967 Jun;33(3):C7–C12. doi: 10.1083/jcb.33.3.c7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Risek B., Guthrie S., Kumar N., Gilula N. B. Modulation of gap junction transcript and protein expression during pregnancy in the rat. J Cell Biol. 1990 Feb;110(2):269–282. doi: 10.1083/jcb.110.2.269. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Unwin P. N., Zampighi G. Structure of the junction between communicating cells. Nature. 1980 Feb 7;283(5747):545–549. doi: 10.1038/283545a0. [DOI] [PubMed] [Google Scholar]
  28. Zimmer D. B., Green C. R., Evans W. H., Gilula N. B. Topological analysis of the major protein in isolated intact rat liver gap junctions and gap junction-derived single membrane structures. J Biol Chem. 1987 Jun 5;262(16):7751–7763. [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES