Biochemical Analysis of Connexon Assembly

  • Judy K. Van Slyke
  • Linda S. Musil
Part of the Methods In Molecular Biology™ book series (MIMB, volume 154)


The formation of gap junctions is a multistep process that begins with synthesis in the membrane of the endoplasmic reticulum (ER) of connexins (Cx), members of a highly homologous family of polytopic integral membrane proteins (see Fig. 1, no. 1) (1). The first stage in gap junction assembly is the noncovalent oligomerization of six connexin monomers into an annular struc ture known as a connexon or hemichannel (see Fig. 1, no. 2). In many (2,3) but apparently not all (4) situations, this event takes place after exit from the ER within an intracellular compartment that is most likely the trans-Golgi network. The connexon complex is then transported to the cell surface (see Fig. 1, no. 3), where it associates head-to-head with a connexon on the surface of an adjacent cell to form an intercellular channel (see Fig. 1,no. 4). Such channels then cluster at up to 10,000/μm2 to form gap junctional “plaques,” the endpoint in gap junction assembly (see Fig. 1, no. 5). An emerging concept is that connexon assembly is not simply a spontaneous default process but instead shows a high degree of selectivity, as evidenced by the finding that only certain types of connexins can co-oligomerize to form heteromeric connexons (5) and the ability of osteoblastic cells to assemble endogenously expressed Cx43, but not Cx46 (3). Determining the mechanism and regulation of connexon formation is therefore a fundamental part of understanding gap junction biosynthesis and function.
Fig. 1.

Schematic of multistep assembly of gap junctional plaques. Assembly begins with the biosynthesis of integral membrane proteins of the connexin family (1). Multiple connexins oligomerize intracellularly to form a connexon (also known as hemichannel) (2) prior to delivery to the plasma membrane (3). Two connexons on apposing cell surfaces associate to form an intercellular channel (4) and channels then cluster into tightly packed arrays classified as gap junctional plaques (5).


Sucrose Gradient Incubation Buffer Tissue Culture Cell Lens Fiber Cell Swing Bucket Rotor 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


  1. 1.
    Goodenough D. A., Goliger J. A., and Paul D. L. (1996) Connexins, connexons, and intercellular communication. Annu. Rev. Biochem. 65, 475–502.PubMedCrossRefGoogle Scholar
  2. 2.
    Musil L., S. and Goodenough D. A. (1993) Multisubunit assembly of an integral plasma membrane channel protein, gap junction connexin43, occurs after exit from the ER. Cell 74, 1065–1077.PubMedCrossRefGoogle Scholar
  3. 3.
    Koval M., Harley J. E., Hick E., and Steinberg T. H. (1997) Connexin46 is retained as monomers in a trans-Golgi compartment of osteoblastic cells. J. Cell Biol. 137, 847–857.PubMedCrossRefGoogle Scholar
  4. 4.
    Kumar N.M, Friend D. S., and Gilula N. B. (1995) Synthesis and assembly of human B1 gap junctions in BHK cells by DNA transfection with the human B1 cDNA.J. Cell Set 108, 3725–3734.Google Scholar
  5. 5.
    Jiang J. X. and Goodenough D. A. (1996) Heteromeric connexons in lens gap junction channels. Proc. Natl. Acad. Sci. USA, 93, 1287–1291.PubMedCrossRefGoogle Scholar
  6. 6.
    Musil L. S. and Goodenough D. A. (1991) Biochemical analysis of connexin43 intracellular transport, phosphorylation, and assembly into gap junctional plaques. J. Cell Biol. 115, 1357–1374.PubMedCrossRefGoogle Scholar
  7. 7.
    Kistler J., Goldie K., Donaldson P., and Engel A. (1994) Reconstitution of native-type noncrystalline lens fiber gap junctions from isolated hemichannels. J. Cell Biol. 126, 1047–1058.PubMedCrossRefGoogle Scholar
  8. 8.
    Stauffer K. A., Kumar N. M., Gilula N. B., and Unwin N. (1991) Isolation and purification of gap junction channels. J. Cell Biol. 115, 141–150.PubMedCrossRefGoogle Scholar
  9. 9.
    Cascio M., Kumar N. M., Safarik R., and Gilula N. B. (1995) Physical characterization of gap junction membrane connexons (hemi-channels) isolated from rat iver.J. Biol. Chem. 270, 18,643–18,648.PubMedCrossRefGoogle Scholar
  10. 10.
    Blount P. and Merlie J. P. (1988) Native folding of the acetylcholine receptor a subunit expressed in the absence of other receptor subunits. J. Biol. Chem. 263, 1072–1080.PubMedGoogle Scholar
  11. 11.
    Musil L. S., Cunningham B. A., Edelman G. M., and Goodenough D. A. (1990) Differential phosphorylation of the gap junction protein connexin43 in junctional communication-competent and-deficient cell lines. J. Cell Biol. 111, 2077–2088.PubMedCrossRefGoogle Scholar
  12. 12.
    Wessel D. and Flugge U. I. (1984) A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids. Anal. Biochem. 138, 141–143.PubMedCrossRefGoogle Scholar
  13. 13.
    Matlin K. S. and Simons K. (1983) Reduced temperature prevents transfer of a membrane glycoprotein to the cell surface but does not prevent terminal glycosylation. Cell 4, 233–243.CrossRefGoogle Scholar
  14. 14.
    Harlow E. and Lane D. (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.Google Scholar
  15. 15.
    Hames B. D. and Rickwood D., ed. (1990) Gel Electrophoresis of Proteins. A Practical Approach, IRL Press, Oxford, England.Google Scholar
  16. 16.
    Towbin H., Staehelin T., and Gordon J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76, 4350–4354.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 2001

Authors and Affiliations

  • Judy K. Van Slyke
    • 1
  • Linda S. Musil
    • 1
  1. 1.Vollum InstituteOregon Health Sciences UniversityPortland

Personalised recommendations