Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 2002 Aug 1;365(Pt 3):693–699. doi: 10.1042/BJ20011572

Post-translational integration and oligomerization of connexin 26 in plasma membranes and evidence of formation of membrane pores: implications for the assembly of gap junctions.

Shoeb Ahmad 1, W Howard Evans 1
PMCID: PMC1222727  PMID: 11985493

Abstract

Gap-junction channels provide a widespread intercellular signalling mechanism. They are constructed of a family of connexin membrane proteins that thread across the membrane four times and oligomerize to generate hexameric gap-junction hemichannels. Using an in vitro cell-free transcription/translation system, we demonstrate that connexin (Cx) 26, one of the smallest connexins, is integrated directly in a post-translational manner into plasma membranes. Protein-cleavage studies of Cx26 integrated into plasma membranes indicate a similar native transmembrane topography to that of Cx26 integrated co-translationally into microsomes. Cx26 integrated post-translationally into plasma membranes oligomerizes and, when incorporated into liposomes, provides permeability to ascorbic acid, suggesting that gap-junction hemichannels are generated. The results provide the basis of a novel alternative mechanism for spontaneous assembly in plasma membranes of Cx26 gap-junction hemichannels that occurs independently of the conventional biogenesis of gap junctions involving connexin trafficking and oligomerization via membrane components of the secretory pathway.

Full Text

The Full Text of this article is available as a PDF (216.1 KB).

Selected References

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

  1. Ahmad S., Diez J. A., George C. H., Evans W. H. Synthesis and assembly of connexins in vitro into homomeric and heteromeric functional gap junction hemichannels. Biochem J. 1999 Apr 15;339(Pt 2):247–253. [PMC free article] [PubMed] [Google Scholar]
  2. Ahmad S., Martin P. E., Evans W. H. Assembly of gap junction channels: mechanism, effects of calmodulin antagonists and identification of connexin oligomerization determinants. Eur J Biochem. 2001 Aug;268(16):4544–4552. doi: 10.1046/j.1432-1327.2001.02380.x. [DOI] [PubMed] [Google Scholar]
  3. Bochkareva E., Seluanov A., Bibi E., Girshovich A. Chaperonin-promoted post-translational membrane insertion of a multispanning membrane protein lactose permease. J Biol Chem. 1996 Sep 6;271(36):22256–22261. doi: 10.1074/jbc.271.36.22256. [DOI] [PubMed] [Google Scholar]
  4. Bruzzone R., White T. W., Paul D. L. Connections with connexins: the molecular basis of direct intercellular signaling. Eur J Biochem. 1996 May 15;238(1):1–27. doi: 10.1111/j.1432-1033.1996.0001q.x. [DOI] [PubMed] [Google Scholar]
  5. Dalbey R. E., Kuhn A. Evolutionarily related insertion pathways of bacterial, mitochondrial, and thylakoid membrane proteins. Annu Rev Cell Dev Biol. 2000;16:51–87. doi: 10.1146/annurev.cellbio.16.1.51. [DOI] [PubMed] [Google Scholar]
  6. Diez J. A., Ahmad S., Evans W. H. Assembly of heteromeric connexons in guinea-pig liver en route to the Golgi apparatus, plasma membrane and gap junctions. Eur J Biochem. 1999 May;262(1):142–148. doi: 10.1046/j.1432-1327.1999.00343.x. [DOI] [PubMed] [Google Scholar]
  7. Evans W. H., Gurd J. W. Preparation and properties of nexuses and lipid-enriched vesicles from mouse liver plasma membranes. Biochem J. 1972 Jul;128(3):691–700. doi: 10.1042/bj1280691. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Falk M. M., Buehler L. K., Kumar N. M., Gilula N. B. Cell-free synthesis and assembly of connexins into functional gap junction membrane channels. EMBO J. 1997 May 15;16(10):2703–2716. doi: 10.1093/emboj/16.10.2703. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Falk M. M., Gilula N. B. Connexin membrane protein biosynthesis is influenced by polypeptide positioning within the translocon and signal peptidase access. J Biol Chem. 1998 Apr 3;273(14):7856–7864. doi: 10.1074/jbc.273.14.7856. [DOI] [PubMed] [Google Scholar]
  10. Foote C. I., Zhou L., Zhu X., Nicholson B. J. The pattern of disulfide linkages in the extracellular loop regions of connexin 32 suggests a model for the docking interface of gap junctions. J Cell Biol. 1998 Mar 9;140(5):1187–1197. doi: 10.1083/jcb.140.5.1187. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. George C. H., Kendall J. M., Campbell A. K., Evans W. H. Connexin-aequorin chimerae report cytoplasmic calcium environments along trafficking pathways leading to gap junction biogenesis in living COS-7 cells. J Biol Chem. 1998 Nov 6;273(45):29822–29829. doi: 10.1074/jbc.273.45.29822. [DOI] [PubMed] [Google Scholar]
  12. George C. H., Kendall J. M., Evans W. H. Intracellular trafficking pathways in the assembly of connexins into gap junctions. J Biol Chem. 1999 Mar 26;274(13):8678–8685. doi: 10.1074/jbc.274.13.8678. [DOI] [PubMed] [Google Scholar]
  13. Goodenough D. A., Goliger J. A., Paul D. L. Connexins, connexons, and intercellular communication. Annu Rev Biochem. 1996;65:475–502. doi: 10.1146/annurev.bi.65.070196.002355. [DOI] [PubMed] [Google Scholar]
  14. Ii M., Mihara K. Insertion of mitochondrial DNA-encoded F1F0-ATPase subunit 8 across the mitochondrial inner membrane in vitro. J Biol Chem. 2001 Apr 24;276(27):24704–24712. doi: 10.1074/jbc.M102584200. [DOI] [PubMed] [Google Scholar]
  15. Jäntti J., Keränen S., Toikkanen J., Kuismanen E., Ehnholm C., Söderlund H., Olkkonen V. M. Membrane insertion and intracellular transport of yeast syntaxin Sso2p in mammalian cells. J Cell Sci. 1994 Dec;107(Pt 12):3623–3633. doi: 10.1242/jcs.107.12.3623. [DOI] [PubMed] [Google Scholar]
  16. Kiselyov K. I., Shin D. M., Wang Y., Pessah I. N., Allen P. D., Muallem S. Gating of store-operated channels by conformational coupling to ryanodine receptors. Mol Cell. 2000 Aug;6(2):421–431. doi: 10.1016/s1097-2765(00)00041-1. [DOI] [PubMed] [Google Scholar]
  17. Kojima T., Fort A., Tao M., Yamamoto M., Spray D. C. Gap junction expression and cell proliferation in differentiating cultures of Cx43 KO mouse hepatocytes. Am J Physiol Gastrointest Liver Physiol. 2001 Oct;281(4):G1004–G1013. doi: 10.1152/ajpgi.2001.281.4.G1004. [DOI] [PubMed] [Google Scholar]
  18. Kojima T., Kokai Y., Chiba H., Yamamoto M., Mochizuki Y., Sawada N. Cx32 but not Cx26 is associated with tight junctions in primary cultures of rat hepatocytes. Exp Cell Res. 2001 Feb 15;263(2):193–201. doi: 10.1006/excr.2000.5103. [DOI] [PubMed] [Google Scholar]
  19. Kojima T., Mitaka T., Shibata Y., Mochizuki Y. Induction and regulation of connexin26 by glucagon in primary cultures of adult rat hepatocytes. J Cell Sci. 1995 Aug;108(Pt 8):2771–2780. doi: 10.1242/jcs.108.8.2771. [DOI] [PubMed] [Google Scholar]
  20. Kojima T., Srinivas M., Fort A., Hopperstad M., Urban M., Hertzberg E. L., Mochizuki Y., Spray D. C. TPA induced expression and function of human connexin 26 by post-translational mechanisms in stably transfected neuroblastoma cells. Cell Struct Funct. 1999 Dec;24(6):435–441. doi: 10.1247/csf.24.435. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. Locke D., Perusinghe N., Newman T., Jayatilake H., Evans W. H., Monaghan P. Developmental expression and assembly of connexins into homomeric and heteromeric gap junction hemichannels in the mouse mammary gland. J Cell Physiol. 2000 May;183(2):228–237. doi: 10.1002/(SICI)1097-4652(200005)183:2<228::AID-JCP9>3.0.CO;2-Y. [DOI] [PubMed] [Google Scholar]
  23. Malewicz B., Kumar V. V., Johnson R. G., Baumann W. J. Lipids in gap junction assembly and function. Lipids. 1990 Aug;25(8):419–427. doi: 10.1007/BF02538083. [DOI] [PubMed] [Google Scholar]
  24. Martin P. E., Blundell G., Ahmad S., Errington R. J., Evans W. H. Multiple pathways in the trafficking and assembly of connexin 26, 32 and 43 into gap junction intercellular communication channels. J Cell Sci. 2001 Nov;114(Pt 21):3845–3855. doi: 10.1242/jcs.114.21.3845. [DOI] [PubMed] [Google Scholar]
  25. Martin P. E., Mambetisaeva E. T., Archer D. A., George C. H., Evans W. H. Analysis of gap junction assembly using mutated connexins detected in Charcot-Marie-Tooth X-linked disease. J Neurochem. 2000 Feb;74(2):711–720. doi: 10.1046/j.1471-4159.2000.740711.x. [DOI] [PubMed] [Google Scholar]
  26. Miles G., Cheley S., Braha O., Bayley H. The staphylococcal leukocidin bicomponent toxin forms large ionic channels. Biochemistry. 2001 Jul 24;40(29):8514–8522. doi: 10.1021/bi010454o. [DOI] [PubMed] [Google Scholar]
  27. Monaghan P., Perusinghe N., Carlile G., Evans W. H. Rapid modulation of gap junction expression in mouse mammary gland during pregnancy, lactation, and involution. J Histochem Cytochem. 1994 Jul;42(7):931–938. doi: 10.1177/42.7.8014476. [DOI] [PubMed] [Google Scholar]
  28. Mueckler M., Lodish H. F. Post-translational insertion of a fragment of the glucose transporter into microsomes requires phosphoanhydride bond cleavage. Nature. 1986 Aug 7;322(6079):549–552. doi: 10.1038/322549a0. [DOI] [PubMed] [Google Scholar]
  29. Nadarajah B., Jones A. M., Evans W. H., Parnavelas J. G. Differential expression of connexins during neocortical development and neuronal circuit formation. J Neurosci. 1997 May 1;17(9):3096–3111. doi: 10.1523/JNEUROSCI.17-09-03096.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Putney J. W., Jr "Kissin' cousins": intimate plasma membrane-ER interactions underlie capacitative calcium entry. Cell. 1999 Oct 1;99(1):5–8. doi: 10.1016/s0092-8674(00)80056-2. [DOI] [PubMed] [Google Scholar]
  31. Rahman S., Carlile G., Evans W. H. Assembly of hepatic gap junctions. Topography and distribution of connexin 32 in intracellular and plasma membranes determined using sequence-specific antibodies. J Biol Chem. 1993 Jan 15;268(2):1260–1265. [PubMed] [Google Scholar]
  32. Rahman S., Evans W. H. Topography of connexin32 in rat liver gap junctions. Evidence for an intramolecular disulphide linkage connecting the two extracellular peptide loops. J Cell Sci. 1991 Nov;100(Pt 3):567–578. doi: 10.1242/jcs.100.3.567. [DOI] [PubMed] [Google Scholar]
  33. Rapoport T. A., Jungnickel B., Kutay U. Protein transport across the eukaryotic endoplasmic reticulum and bacterial inner membranes. Annu Rev Biochem. 1996;65:271–303. doi: 10.1146/annurev.bi.65.070196.001415. [DOI] [PubMed] [Google Scholar]
  34. Sakaguchi M., Hachiya N., Mihara K., Omura T. Mitochondrial porin can be translocated across both endoplasmic reticulum and mitochondrial membranes. J Biochem. 1992 Aug;112(2):243–248. doi: 10.1093/oxfordjournals.jbchem.a123884. [DOI] [PubMed] [Google Scholar]
  35. Unger V. M., Kumar N. M., Gilula N. B., Yeager M. Three-dimensional structure of a recombinant gap junction membrane channel. Science. 1999 Feb 19;283(5405):1176–1180. doi: 10.1126/science.283.5405.1176. [DOI] [PubMed] [Google Scholar]
  36. Valeva A., Schnabel R., Walev I., Boukhallouk F., Bhakdi S., Palmer M. Membrane insertion of the heptameric staphylococcal alpha-toxin pore. A domino-like structural transition that is allosterically modulated by the target cell membrane. J Biol Chem. 2001 Feb 2;276(18):14835–14841. doi: 10.1074/jbc.M100301200. [DOI] [PubMed] [Google Scholar]
  37. Zhang J. T., Chen M., Foote C. I., Nicholson B. J. Membrane integration of in vitro-translated gap junctional proteins: co- and post-translational mechanisms. Mol Biol Cell. 1996 Mar;7(3):471–482. doi: 10.1091/mbc.7.3.471. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Zhang J. T., Nicholson B. J. The topological structure of connexin 26 and its distribution compared to connexin 32 in hepatic gap junctions. J Membr Biol. 1994 Apr;139(1):15–29. doi: 10.1007/BF00232671. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES