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. 1996 Dec;7(12):1995–2006. doi: 10.1091/mbc.7.12.1995

Incompatibility of connexin 40 and 43 Hemichannels in gap junctions between mammalian cells is determined by intracellular domains.

S Haubrich 1, H J Schwarz 1, F Bukauskas 1, H Lichtenberg-Fraté 1, O Traub 1, R Weingart 1, K Willecke 1
PMCID: PMC276045  PMID: 8970160

Abstract

Murine connexin 40 (Cx40) and connexin 43 (Cx43) do not form functional heterotypic gap junction channels. This property may contribute to the preferential propagation of action potentials in murine conductive myocardium (expressing Cx40) which is surrounded by working myocardium, expressing Cx43. When mouse Cx40 and Cx43 were individually expressed in cocultured human HeLa cells, no punctate immunofluorescent signals were detected on apposed plasma membranes between different transfectants, using antibodies specific for each connexin, suggesting that Cx40 and Cx43 hemichannels do not dock to each other. We wanted to identify domains in these connexin proteins which are responsible for the incompatibility. Thus, we expressed in HeLa cells several chimeric gene constructs in which different extracellular and intracellular domains of Cx43 had been spliced into the corresponding regions of Cx40. We found that exchange of both extracellular loops (E1 and E2) in this system (Cx40*43E1,2) was required for formation of homotypic and heterotypic conductive channels, although the electrical properties differed from those of Cx40 or Cx43 channels. Thus, the extracellular domains of Cx43 can be directed to form functional homo- and heterotypic channels. Another chimeric construct in which both extracellular domains and the central cytoplasmic loop (E1, E2, and C2) of Cx43 were spliced into Cx40 (Cx40*43E1,2,C2) led to heterotypic coupling only with Cx43 and not with Cx40 transfectants. Thus, the central cytoplasmic loop of Cx43 contributed to selectivity. A third construct, in which only the C-terminal domain (C3) of Cx43 was spliced into Cx40, i.e., Cx40*43C3, showed neither homotypic nor heterotypic coupling with Cx40 and Cx43 transfectants, suggesting that the C-terminal region of Cx43 determined incompatibility.

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1995

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Selected References

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  1. Bastide B., Neyses L., Ganten D., Paul M., Willecke K., Traub O. Gap junction protein connexin40 is preferentially expressed in vascular endothelium and conductive bundles of rat myocardium and is increased under hypertensive conditions. Circ Res. 1993 Dec;73(6):1138–1149. doi: 10.1161/01.res.73.6.1138. [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. Bruzzone R., Haefliger J. A., Gimlich R. L., Paul D. L. Connexin40, a component of gap junctions in vascular endothelium, is restricted in its ability to interact with other connexins. Mol Biol Cell. 1993 Jan;4(1):7–20. doi: 10.1091/mbc.4.1.7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chen C., Okayama H. High-efficiency transformation of mammalian cells by plasmid DNA. Mol Cell Biol. 1987 Aug;7(8):2745–2752. doi: 10.1128/mcb.7.8.2745. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  6. Clackson T., Winter G. 'Sticky feet'-directed mutagenesis and its application to swapping antibody domains. Nucleic Acids Res. 1989 Dec 25;17(24):10163–10170. doi: 10.1093/nar/17.24.10163. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Dahl G., Nonner W., Werner R. Attempts to define functional domains of gap junction proteins with synthetic peptides. Biophys J. 1994 Nov;67(5):1816–1822. doi: 10.1016/S0006-3495(94)80663-0. [DOI] [PMC free article] [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. Dermietzel R., Yancey B., Janssen-Timmen U., Traub O., Willecke K., Revel J. P. Simultaneous light and electron microscopic observation of immunolabeled liver 27 KD gap junction protein on ultra-thin cryosections. J Histochem Cytochem. 1987 Mar;35(3):387–392. doi: 10.1177/35.3.3029214. [DOI] [PubMed] [Google Scholar]
  10. Eckert R., Dunina-Barkovskaya A., Hülser D. F. Biophysical characterization of gap-junction channels in HeLa cells. Pflugers Arch. 1993 Aug;424(3-4):335–342. doi: 10.1007/BF00384361. [DOI] [PubMed] [Google Scholar]
  11. Elfgang C., Eckert R., Lichtenberg-Fraté H., Butterweck A., Traub O., Klein R. A., Hülser D. F., Willecke K. Specific permeability and selective formation of gap junction channels in connexin-transfected HeLa cells. J Cell Biol. 1995 May;129(3):805–817. doi: 10.1083/jcb.129.3.805. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gros D., Jarry-Guichard T., Ten Velde I., de Maziere A., van Kempen M. J., Davoust J., Briand J. P., Moorman A. F., Jongsma H. J. Restricted distribution of connexin40, a gap junctional protein, in mammalian heart. Circ Res. 1994 May;74(5):839–851. doi: 10.1161/01.res.74.5.839. [DOI] [PubMed] [Google Scholar]
  13. Haefliger J. A., Bruzzone R., Jenkins N. A., Gilbert D. J., Copeland N. G., Paul D. L. Four novel members of the connexin family of gap junction proteins. Molecular cloning, expression, and chromosome mapping. J Biol Chem. 1992 Jan 25;267(3):2057–2064. [PubMed] [Google Scholar]
  14. Hennemann H., Suchyna T., Lichtenberg-Fraté H., Jungbluth S., Dahl E., Schwarz J., Nicholson B. J., Willecke K. Molecular cloning and functional expression of mouse connexin40, a second gap junction gene preferentially expressed in lung. J Cell Biol. 1992 Jun;117(6):1299–1310. doi: 10.1083/jcb.117.6.1299. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hertzberg E. L., Disher R. M., Tiller A. A., Zhou Y., Cook R. G. Topology of the Mr 27,000 liver gap junction protein. Cytoplasmic localization of amino- and carboxyl termini and a hydrophilic domain which is protease-hypersensitive. J Biol Chem. 1988 Dec 15;263(35):19105–19111. [PubMed] [Google Scholar]
  16. Horst M., Harth N., Hasilik A. Biosynthesis of glycosylated human lysozyme mutants. J Biol Chem. 1991 Jul 25;266(21):13914–13919. [PubMed] [Google Scholar]
  17. Kramer W., Fritz H. J. Oligonucleotide-directed construction of mutations via gapped duplex DNA. Methods Enzymol. 1987;154:350–367. doi: 10.1016/0076-6879(87)54084-8. [DOI] [PubMed] [Google Scholar]
  18. Kumar N. M., Gilula N. B. The gap junction communication channel. Cell. 1996 Feb 9;84(3):381–388. doi: 10.1016/s0092-8674(00)81282-9. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. 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]
  21. 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]
  22. Norton P. A. Alternative pre-mRNA splicing: factors involved in splice site selection. J Cell Sci. 1994 Jan;107(Pt 1):1–7. doi: 10.1242/jcs.107.1.1. [DOI] [PubMed] [Google Scholar]
  23. Paul D. L. New functions for gap junctions. Curr Opin Cell Biol. 1995 Oct;7(5):665–672. doi: 10.1016/0955-0674(95)80108-1. [DOI] [PubMed] [Google Scholar]
  24. Rubin J. B., Verselis V. K., Bennett M. V., Bargiello T. A. A domain substitution procedure and its use to analyze voltage dependence of homotypic gap junctions formed by connexins 26 and 32. Proc Natl Acad Sci U S A. 1992 May 1;89(9):3820–3824. doi: 10.1073/pnas.89.9.3820. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Traub O., Eckert R., Lichtenberg-Fraté H., Elfgang C., Bastide B., Scheidtmann K. H., Hülser D. F., Willecke K. Immunochemical and electrophysiological characterization of murine connexin40 and -43 in mouse tissues and transfected human cells. Eur J Cell Biol. 1994 Jun;64(1):101–112. [PubMed] [Google Scholar]
  27. Warner A., Clements D. K., Parikh S., Evans W. H., DeHaan R. L. Specific motifs in the external loops of connexin proteins can determine gap junction formation between chick heart myocytes. J Physiol. 1995 Nov 1;488(Pt 3):721–728. doi: 10.1113/jphysiol.1995.sp021003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Weingart R. Electrical properties of the nexal membrane studied in rat ventricular cell pairs. J Physiol. 1986 Jan;370:267–284. doi: 10.1113/jphysiol.1986.sp015934. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. White T. W., Bruzzone R., Paul D. L. The connexin family of intercellular channel forming proteins. Kidney Int. 1995 Oct;48(4):1148–1157. doi: 10.1038/ki.1995.398. [DOI] [PubMed] [Google Scholar]
  30. White T. W., Bruzzone R., Wolfram S., Paul D. L., Goodenough D. A. Selective interactions among the multiple connexin proteins expressed in the vertebrate lens: the second extracellular domain is a determinant of compatibility between connexins. J Cell Biol. 1994 May;125(4):879–892. doi: 10.1083/jcb.125.4.879. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. White T. W., Paul D. L., Goodenough D. A., Bruzzone R. Functional analysis of selective interactions among rodent connexins. Mol Biol Cell. 1995 Apr;6(4):459–470. doi: 10.1091/mbc.6.4.459. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Willecke K., Heynkes R., Dahl E., Stutenkemper R., Hennemann H., Jungbluth S., Suchyna T., Nicholson B. J. Mouse connexin37: cloning and functional expression of a gap junction gene highly expressed in lung. J Cell Biol. 1991 Sep;114(5):1049–1057. doi: 10.1083/jcb.114.5.1049. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Yancey S. B., John S. A., Lal R., Austin B. J., Revel J. P. The 43-kD polypeptide of heart gap junctions: immunolocalization, topology, and functional domains. J Cell Biol. 1989 Jun;108(6):2241–2254. doi: 10.1083/jcb.108.6.2241. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Yeager M., Gilula N. B. Membrane topology and quaternary structure of cardiac gap junction ion channels. J Mol Biol. 1992 Feb 20;223(4):929–948. doi: 10.1016/0022-2836(92)90253-g. [DOI] [PubMed] [Google Scholar]
  35. 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]

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