Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1987 Dec 1;105(6):2621–2629. doi: 10.1083/jcb.105.6.2621

Connexin43: a protein from rat heart homologous to a gap junction protein from liver

PMCID: PMC2114703  PMID: 2826492

Abstract

Northern blot analysis of rat heart mRNA probed with a cDNA coding for the principal polypeptide of rat liver gap junctions demonstrated a 3.0- kb band. This band was observed only after hybridization and washing using low stringency conditions; high stringency conditions abolished the hybridization. A rat heart cDNA library was screened with the same cDNA probe under the permissive hybridization conditions, and a single positive clone identified and purified. The clone contained a 220-bp insert, which showed 55% homology to the original cDNA probe near the 5' end. The 220-bp cDNA was used to rescreen a heart cDNA library under high stringency conditions, and three additional cDNAs that together spanned 2,768 bp were isolated. This composite cDNA contained a single 1,146-bp open reading frame coding for a predicted polypeptide of 382 amino acids with a molecular mass of 43,036 D. Northern analysis of various rat tissues using this heart cDNA as probe showed hybridization to 3.0-kb bands in RNA isolated from heart, ovary, uterus, kidney, and lens epithelium. Comparisons of the predicted amino acid sequences for the two gap junction proteins isolated from heart and liver showed two regions of high homology (58 and 42%), and other regions of little or no homology. A model is presented which indicates that the conserved sequences correspond to transmembrane and extracellular regions of the junctional molecules, while the nonconserved sequences correspond to cytoplasmic regions. Since it has been shown previously that the original cDNA isolated from liver recognizes mRNAs in stomach, kidney, and brain, and it is shown here that the cDNA isolated from heart recognizes mRNAs in ovary, uterus, lens epithelium, and kidney, a nomenclature is proposed which avoids categorization by organ of origin. In this nomenclature, the homologous proteins in gap junctions would be called connexins, each distinguished by its predicted molecular mass in kilodaltons. The gap junction protein isolated from liver would then be called connexin32; from heart, connexin43.

Full Text

The Full Text of this article is available as a PDF (1.3 MB).

Selected References

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

  1. Albertini D. F., Anderson E. The appearance and structure of intercellular connections during the ontogeny of the rabbit ovarian follicle with particular reference to gap junctions. J Cell Biol. 1974 Oct;63(1):234–250. doi: 10.1083/jcb.63.1.234. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Atkinson M. M., Menko A. S., Johnson R. G., Sheppard J. R., Sheridan J. D. Rapid and reversible reduction of junctional permeability in cells infected with a temperature-sensitive mutant of avian sarcoma virus. J Cell Biol. 1981 Nov;91(2 Pt 1):573–578. doi: 10.1083/jcb.91.2.573. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Aviv H., Leder P. Purification of biologically active globin messenger RNA by chromatography on oligothymidylic acid-cellulose. Proc Natl Acad Sci U S A. 1972 Jun;69(6):1408–1412. doi: 10.1073/pnas.69.6.1408. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Azarnia R., Loewenstein W. R. Polyomavirus middle T antigen downregulates junctional cell-to-cell communication. Mol Cell Biol. 1987 Feb;7(2):946–950. doi: 10.1128/mcb.7.2.946. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Brewer G. J., Thomas P. D. Role of gangliosides in adhesion and conductance changes in large spherical model membranes. Biochim Biophys Acta. 1984 Oct 3;776(2):279–287. doi: 10.1016/0005-2736(84)90217-7. [DOI] [PubMed] [Google Scholar]
  6. Chirgwin J. M., Przybyla A. E., MacDonald R. J., Rutter W. J. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry. 1979 Nov 27;18(24):5294–5299. doi: 10.1021/bi00591a005. [DOI] [PubMed] [Google Scholar]
  7. Dahl G., Miller T., Paul D., Voellmy R., Werner R. Expression of functional cell-cell channels from cloned rat liver gap junction complementary DNA. Science. 1987 Jun 5;236(4806):1290–1293. doi: 10.1126/science.3035715. [DOI] [PubMed] [Google Scholar]
  8. Dermietzel R., Leibstein A., Frixen U., Janssen-Timmen U., Traub O., Willecke K. Gap junctions in several tissues share antigenic determinants with liver gap junctions. EMBO J. 1984 Oct;3(10):2261–2270. doi: 10.1002/j.1460-2075.1984.tb02124.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Fallon R. F., Goodenough D. A. Five-hour half-life of mouse liver gap-junction protein. J Cell Biol. 1981 Aug;90(2):521–526. doi: 10.1083/jcb.90.2.521. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Feinberg A. P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
  11. FitzGerald P. G., Goodenough D. A. Rat lens cultures: MIP expression and domains of intercellular coupling. Invest Ophthalmol Vis Sci. 1986 May;27(5):755–771. [PubMed] [Google Scholar]
  12. Flagg-Newton J., Loewenstein W. R. Experimental depression of junctional membrane permeability in mammalian cell culture. A study with tracer molecules in the 300 to 800 Dalton range. J Membr Biol. 1979 Oct 5;50(1):65–100. doi: 10.1007/BF01868788. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Fraser S. E., Green C. R., Bode H. R., Gilula N. B. Selective disruption of gap junctional communication interferes with a patterning process in hydra. Science. 1987 Jul 3;237(4810):49–55. doi: 10.1126/science.3037697. [DOI] [PubMed] [Google Scholar]
  14. Garfield R. E., Daniel E. E., Dukes M., Fitzgerald J. D. Changes of gap junctions in myometrium of guinea pig at parturition and abortion. Can J Physiol Pharmacol. 1982 Mar;60(3):335–341. doi: 10.1139/y82-047. [DOI] [PubMed] [Google Scholar]
  15. Gorin M. B., Yancey S. B., Cline J., Revel J. P., Horwitz J. The major intrinsic protein (MIP) of the bovine lens fiber membrane: characterization and structure based on cDNA cloning. Cell. 1984 Nov;39(1):49–59. doi: 10.1016/0092-8674(84)90190-9. [DOI] [PubMed] [Google Scholar]
  16. Gubler U., Hoffman B. J. A simple and very efficient method for generating cDNA libraries. Gene. 1983 Nov;25(2-3):263–269. doi: 10.1016/0378-1119(83)90230-5. [DOI] [PubMed] [Google Scholar]
  17. Helms C., Graham M. Y., Dutchik J. E., Olson M. V. A new method for purifying lambda DNA from phage lysates. DNA. 1985 Feb;4(1):39–49. doi: 10.1089/dna.1985.4.39. [DOI] [PubMed] [Google Scholar]
  18. Henikoff S. Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing. Gene. 1984 Jun;28(3):351–359. doi: 10.1016/0378-1119(84)90153-7. [DOI] [PubMed] [Google Scholar]
  19. Hertzberg E. L., Skibbens R. V. A protein homologous to the 27,000 dalton liver gap junction protein is present in a wide variety of species and tissues. Cell. 1984 Nov;39(1):61–69. doi: 10.1016/0092-8674(84)90191-0. [DOI] [PubMed] [Google Scholar]
  20. Hertzberg E. L., Spray D. C., Bennett M. V. Reduction of gap junctional conductance by microinjection of antibodies against the 27-kDa liver gap junction polypeptide. Proc Natl Acad Sci U S A. 1985 Apr;82(8):2412–2416. doi: 10.1073/pnas.82.8.2412. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. 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]
  22. Kyte J., Doolittle R. F. A simple method for displaying the hydropathic character of a protein. J Mol Biol. 1982 May 5;157(1):105–132. doi: 10.1016/0022-2836(82)90515-0. [DOI] [PubMed] [Google Scholar]
  23. Lawler J., Hynes R. O. The structure of human thrombospondin, an adhesive glycoprotein with multiple calcium-binding sites and homologies with several different proteins. J Cell Biol. 1986 Nov;103(5):1635–1648. doi: 10.1083/jcb.103.5.1635. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Manjunath C. K., Nicholson B. J., Teplow D., Hood L., Page E., Revel J. P. The cardiac gap junction protein (Mr 47,000) has a tissue-specific cytoplasmic domain of Mr 17,000 at its carboxy-terminus. Biochem Biophys Res Commun. 1987 Jan 15;142(1):228–234. doi: 10.1016/0006-291x(87)90475-x. [DOI] [PubMed] [Google Scholar]
  25. Manjunath C. K., Page E. Cell biology and protein composition of cardiac gap junctions. Am J Physiol. 1985 Jun;248(6 Pt 2):H783–H791. doi: 10.1152/ajpheart.1985.248.6.H783. [DOI] [PubMed] [Google Scholar]
  26. Milstone L. M., Piatigorsky J. Rates of protein synthesis in explanted embryonic chick lens epithelia: differential stimulation of delta-crystallin synthesis. Dev Biol. 1975 Mar;43(1):91–100. doi: 10.1016/0012-1606(75)90133-5. [DOI] [PubMed] [Google Scholar]
  27. Nicholson B. J., Gros D. B., Kent S. B., Hood L. E., Revel J. P. The Mr 28,000 gap junction proteins from rat heart and liver are different but related. J Biol Chem. 1985 Jun 10;260(11):6514–6517. [PubMed] [Google Scholar]
  28. 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]
  29. Peracchia C., Girsch S. J. Permeability and gating of lens gap junction channels incorporated into liposomes. Curr Eye Res. 1985 Apr;4(4):431–439. doi: 10.3109/02713688509025157. [DOI] [PubMed] [Google Scholar]
  30. Reber W. R., Weingart R. Ungulate cardiac purkinje fibres: the influence of intracellular pH on the electrical cell-to-cell coupling. J Physiol. 1982 Jul;328:87–104. doi: 10.1113/jphysiol.1982.sp014254. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Saez J. C., Spray D. C., Nairn A. C., Hertzberg E., Greengard P., Bennett M. V. cAMP increases junctional conductance and stimulates phosphorylation of the 27-kDa principal gap junction polypeptide. Proc Natl Acad Sci U S A. 1986 Apr;83(8):2473–2477. doi: 10.1073/pnas.83.8.2473. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Spray D. C., Ginzberg R. D., Morales E. A., Gatmaitan Z., Arias I. M. Electrophysiological properties of gap junctions between dissociated pairs of rat hepatocytes. J Cell Biol. 1986 Jul;103(1):135–144. doi: 10.1083/jcb.103.1.135. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Spray D. C., Saez J. C., Brosius D., Bennett M. V., Hertzberg E. L. Isolated liver gap junctions: gating of transjunctional currents is similar to that in intact pairs of rat hepatocytes. Proc Natl Acad Sci U S A. 1986 Aug;83(15):5494–5497. doi: 10.1073/pnas.83.15.5494. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Warner A. E., Guthrie S. C., Gilula N. B. Antibodies to gap-junctional protein selectively disrupt junctional communication in the early amphibian embryo. Nature. 1984 Sep 13;311(5982):127–131. doi: 10.1038/311127a0. [DOI] [PubMed] [Google Scholar]
  35. White R. L., Spray D. C., Campos de Carvalho A. C., Wittenberg B. A., Bennett M. V. Some electrical and pharmacological properties of gap junctions between adult ventricular myocytes. Am J Physiol. 1985 Nov;249(5 Pt 1):C447–C455. doi: 10.1152/ajpcell.1985.249.5.C447. [DOI] [PubMed] [Google Scholar]
  36. Wiener E. C., Loewenstein W. R. Correction of cell-cell communication defect by introduction of a protein kinase into mutant cells. 1983 Sep 29-Oct 5Nature. 305(5933):433–435. doi: 10.1038/305433a0. [DOI] [PubMed] [Google Scholar]
  37. Yancey S. B., Nicholson B. J., Revel J. P. The dynamic state of liver gap junctions. J Supramol Struct Cell Biochem. 1981;16(3):221–232. doi: 10.1002/jsscb.1981.380160303. [DOI] [PubMed] [Google Scholar]
  38. Young J. D., Cohn Z. A., Gilula N. B. Functional assembly of gap junction conductance in lipid bilayers: demonstration that the major 27 kd protein forms the junctional channel. Cell. 1987 Mar 13;48(5):733–743. doi: 10.1016/0092-8674(87)90071-7. [DOI] [PubMed] [Google Scholar]
  39. Zervos A. S., Hope J., Evans W. H. Preparation of a gap junction fraction from uteri of pregnant rats: the 28-kD polypeptides of uterus, liver, and heart gap junctions are homologous. J Cell Biol. 1985 Oct;101(4):1363–1370. doi: 10.1083/jcb.101.4.1363. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. 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