Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 2000 Jul 1;349(Pt 1):281–287. doi: 10.1042/0264-6021:3490281

Targeting motifs and functional parameters governing the assembly of connexins into gap junctions.

P E Martin 1, J Steggles 1, C Wilson 1, S Ahmad 1, W H Evans 1
PMCID: PMC1221149  PMID: 10861240

Abstract

To study the assembly of gap junctions, connexin--green-fluorescent-protein (Cx--GFP) chimeras were expressed in COS-7 and HeLa cells. Cx26-- and Cx32--GFP were targeted to gap junctions where they formed functional channels that transferred Lucifer Yellow. A series of Cx32--GFP chimeras, truncated from the C-terminal cytoplasmic tail, were studied to identify amino acid sequences governing targeting from intracellular assembly sites to the gap junction. Extensive truncation of Cx32 resulted in failure to integrate into membranes. Truncation of Cx32 to residue 207, corresponding to removal of most of the 78 amino acids on the cytoplasmic C-terminal tail, led to arrest in the endoplasmic reticulum and incomplete oligomerization. However, truncation to amino acid 219 did not impair Cx oligomerization and connexon hemichannels were targeted to the plasma membrane. It was concluded that a crucial gap-junction targeting sequence resides between amino acid residues 207 and 219 on the cytoplasmic C-terminal tail of Cx32. Studies of a Cx32E208K mutation identified this as one of the key amino acids dictating targeting to the gap junction, although oligomerization of this site-specific mutation into hexameric hemichannels was relatively unimpaired. The studies show that expression of these Cx--GFP constructs in mammalian cells allowed an analysis of amino acid residues involved in gap-junction assembly.

Full Text

The Full Text of this article is available as a PDF (252.1 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. Bergoffen J., Scherer S. S., Wang S., Scott M. O., Bone L. J., Paul D. L., Chen K., Lensch M. W., Chance P. F., Fischbeck K. H. Connexin mutations in X-linked Charcot-Marie-Tooth disease. Science. 1993 Dec 24;262(5142):2039–2042. doi: 10.1126/science.8266101. [DOI] [PubMed] [Google Scholar]
  3. Cascio M., Kumar N. M., Safarik R., Gilula N. B. Physical characterization of gap junction membrane connexons (hemi-channels) isolated from rat liver. J Biol Chem. 1995 Aug 4;270(31):18643–18648. doi: 10.1074/jbc.270.31.18643. [DOI] [PubMed] [Google Scholar]
  4. Castro C., Gómez-Hernandez J. M., Silander K., Barrio L. C. Altered formation of hemichannels and gap junction channels caused by C-terminal connexin-32 mutations. J Neurosci. 1999 May 15;19(10):3752–3760. doi: 10.1523/JNEUROSCI.19-10-03752.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Deschênes S. M., Walcott J. L., Wexler T. L., Scherer S. S., Fischbeck K. H. Altered trafficking of mutant connexin32. J Neurosci. 1997 Dec 1;17(23):9077–9084. doi: 10.1523/JNEUROSCI.17-23-09077.1997. [DOI] [PMC free article] [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. Ek-Vitorín J. F., Calero G., Morley G. E., Coombs W., Taffet S. M., Delmar M. PH regulation of connexin43: molecular analysis of the gating particle. Biophys J. 1996 Sep;71(3):1273–1284. doi: 10.1016/S0006-3495(96)79328-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Evans W. H., Ahmad S., Diez J., George C. H., Kendall J. M., Martin P. E. Trafficking pathways leading to the formation of gap junctions. Novartis Found Symp. 1999;219:44–59. doi: 10.1002/9780470515587.ch4. [DOI] [PubMed] [Google Scholar]
  9. Evans W. H. Assembly of gap junction intercellular communication channels. Biochem Soc Trans. 1994 Aug;22(3):788–792. doi: 10.1042/bst0220788. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Galvan D. L., Borrego-Diaz E., Perez P. J., Mignery G. A. Subunit oligomerization, and topology of the inositol 1,4, 5-trisphosphate receptor. J Biol Chem. 1999 Oct 8;274(41):29483–29492. doi: 10.1074/jbc.274.41.29483. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. 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]
  14. George C. H., Martin P. E., Evans W. H. Rapid determination of gap junction formation using HeLa cells microinjected with cDNAs encoding wild-type and chimeric connexins. Biochem Biophys Res Commun. 1998 Jun 29;247(3):785–789. doi: 10.1006/bbrc.1998.8835. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. He D. S., Jiang J. X., Taffet S. M., Burt J. M. Formation of heteromeric gap junction channels by connexins 40 and 43 in vascular smooth muscle cells. Proc Natl Acad Sci U S A. 1999 May 25;96(11):6495–6500. doi: 10.1073/pnas.96.11.6495. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hopkins W. F., Demas V., Tempel B. L. Both N- and C-terminal regions contribute to the assembly and functional expression of homo- and heteromultimeric voltage-gated K+ channels. J Neurosci. 1994 Mar;14(3 Pt 1):1385–1393. doi: 10.1523/JNEUROSCI.14-03-01385.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Jordan K., Solan J. L., Dominguez M., Sia M., Hand A., Lampe P. D., Laird D. W. Trafficking, assembly, and function of a connexin43-green fluorescent protein chimera in live mammalian cells. Mol Biol Cell. 1999 Jun;10(6):2033–2050. doi: 10.1091/mbc.10.6.2033. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kelsell D. P., Dunlop J., Stevens H. P., Lench N. J., Liang J. N., Parry G., Mueller R. F., Leigh I. M. Connexin 26 mutations in hereditary non-syndromic sensorineural deafness. Nature. 1997 May 1;387(6628):80–83. doi: 10.1038/387080a0. [DOI] [PubMed] [Google Scholar]
  20. Kendall J. M., Badminton M. N. Aequorea victoria bioluminescence moves into an exciting new era. Trends Biotechnol. 1998 May;16(5):216–224. doi: 10.1016/s0167-7799(98)01184-6. [DOI] [PubMed] [Google Scholar]
  21. Martin P. E., Coleman S. L., Casalotti S. O., Forge A., Evans W. H. Properties of connexin26 gap junctional proteins derived from mutations associated with non-syndromal heriditary deafness. Hum Mol Genet. 1999 Dec;8(13):2369–2376. doi: 10.1093/hmg/8.13.2369. [DOI] [PubMed] [Google Scholar]
  22. Martin P. E., George C. H., Castro C., Kendall J. M., Capel J., Campbell A. K., Revilla A., Barrio L. C., Evans W. H. Assembly of chimeric connexin-aequorin proteins into functional gap junction channels. Reporting intracellular and plasma membrane calcium environments. J Biol Chem. 1998 Jan 16;273(3):1719–1726. doi: 10.1074/jbc.273.3.1719. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. Musil L. S., Goodenough D. A. Multisubunit assembly of an integral plasma membrane channel protein, gap junction connexin43, occurs after exit from the ER. Cell. 1993 Sep 24;74(6):1065–1077. doi: 10.1016/0092-8674(93)90728-9. [DOI] [PubMed] [Google Scholar]
  25. Oh S., Ri Y., Bennett M. V., Trexler E. B., Verselis V. K., Bargiello T. A. Changes in permeability caused by connexin 32 mutations underlie X-linked Charcot-Marie-Tooth disease. Neuron. 1997 Oct;19(4):927–938. doi: 10.1016/s0896-6273(00)80973-3. [DOI] [PubMed] [Google Scholar]
  26. Omori Y., Mesnil M., Yamasaki H. Connexin 32 mutations from X-linked Charcot-Marie-Tooth disease patients: functional defects and dominant negative effects. Mol Biol Cell. 1996 Jun;7(6):907–916. doi: 10.1091/mbc.7.6.907. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. 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]
  28. Roderick H. L., Campbell A. K., Llewellyn D. H. Nuclear localisation of calreticulin in vivo is enhanced by its interaction with glucocorticoid receptors. FEBS Lett. 1997 Mar 24;405(2):181–185. doi: 10.1016/s0014-5793(97)00183-x. [DOI] [PubMed] [Google Scholar]
  29. Shiels A., Mackay D., Ionides A., Berry V., Moore A., Bhattacharya S. A missense mutation in the human connexin50 gene (GJA8) underlies autosomal dominant "zonular pulverulent" cataract, on chromosome 1q. Am J Hum Genet. 1998 Mar;62(3):526–532. doi: 10.1086/301762. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Simon S. Translocation of proteins across the endoplasmic reticulum. Curr Opin Cell Biol. 1993 Aug;5(4):581–588. doi: 10.1016/0955-0674(93)90126-B. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Sullivan R., Lo C. W. Expression of a connexin 43/beta-galactosidase fusion protein inhibits gap junctional communication in NIH3T3 cells. J Cell Biol. 1995 Jul;130(2):419–429. doi: 10.1083/jcb.130.2.419. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Tenjinbaru K., Furuno T., Hirashima N., Nakanishi M. Nuclear translocation of green fluorescent protein-nuclear factor kappaB with a distinct lag time in living cells. FEBS Lett. 1999 Feb 5;444(1):1–4. doi: 10.1016/s0014-5793(99)00002-2. [DOI] [PubMed] [Google Scholar]
  33. Trexler E. B., Bennett M. V., Bargiello T. A., Verselis V. K. Voltage gating and permeation in a gap junction hemichannel. Proc Natl Acad Sci U S A. 1996 Jun 11;93(12):5836–5841. doi: 10.1073/pnas.93.12.5836. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Török K., Stauffer K., Evans W. H. Connexin 32 of gap junctions contains two cytoplasmic calmodulin-binding domains. Biochem J. 1997 Sep 1;326(Pt 2):479–483. doi: 10.1042/bj3260479. [DOI] [PMC free article] [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. Wang X., Li L., Peracchia L. L., Peracchia C. Chimeric evidence for a role of the connexin cytoplasmic loop in gap junction channel gating. Pflugers Arch. 1996 Apr;431(6):844–852. doi: 10.1007/s004240050076. [DOI] [PubMed] [Google Scholar]
  37. Xia J. H., Liu C. Y., Tang B. S., Pan Q., Huang L., Dai H. P., Zhang B. R., Xie W., Hu D. X., Zheng D. Mutations in the gene encoding gap junction protein beta-3 associated with autosomal dominant hearing impairment. Nat Genet. 1998 Dec;20(4):370–373. doi: 10.1038/3845. [DOI] [PubMed] [Google Scholar]
  38. Yoshimura T., Satake M., Ohnishi A., Tsutsumi Y., Fujikura Y. Mutations of connexin32 in Charcot-Marie-Tooth disease type X interfere with cell-to-cell communication but not cell proliferation and myelin-specific gene expression. J Neurosci Res. 1998 Jan 15;51(2):154–161. doi: 10.1002/(SICI)1097-4547(19980115)51:2<154::AID-JNR4>3.0.CO;2-C. [DOI] [PubMed] [Google Scholar]
  39. Zhang J. T., Nicholson B. J. Sequence and tissue distribution of a second protein of hepatic gap junctions, Cx26, as deduced from its cDNA. J Cell Biol. 1989 Dec;109(6 Pt 2):3391–3401. doi: 10.1083/jcb.109.6.3391. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES