Skip to main content
NCBI home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1995 May 1;129(3):805–817. doi: 10.1083/jcb.129.3.805

Specific permeability and selective formation of gap junction channels in connexin-transfected HeLa cells

PMCID: PMC2120441  PMID: 7537274

Abstract

DNAs coding for seven murine connexins (Cx) (Cx26, Cx31, Cx32, Cx37, Cx40, Cx43, and Cx45) are functionally expressed in human HeLa cells that were deficient in gap junctional communication. We compare the permeabilities of gap junctions comprised of different connexins to iontophoretically injected tracer molecules. Our results show that Lucifer yellow can pass through all connexin channels analyzed. On the other hand, propidium iodide and ethidium bromide penetrate very poorly or not at all through Cx31 and Cx32 channels, respectively, but pass through channels of other connexins. 4,6 Diamidino-2-phenylindole (DAPI) dihydrochloride shows less transfer among Cx31 or Cx43 transfectants. Neurobiotin is weakly transferred among Cx31 transfectants. Total junctional conductance in Cx31 or Cx45 transfected cells is only about half as high as in other connexin transfectants analyzed and does not correlate exactly with any of the tracer permeabilities. Permeability through different connexin channels appears to be dependent on the molecular structure of each tracer, i.e. size, charge and possibly rigidity. This supports the hypothesis that different connexin channels show different permeabilities to second messenger molecules as well as metabolites and may fulfill in this way their specific role in growth control and differentiation of cell types. In addition, we have investigated the function of heterotypic gap junctions after co-cultivation of two different connexin transfectants, one of which had been prelabeled with fluorescent dextran beads. Analysis of Lucifer yellow transfer reveals that HeLa cells expressing Cx31 (beta-type connexin) do not communicate with any other connexin transfectant tested but only with themselves. Two other beta-type connexin transfectants, HeLa-Cx26 and -Cx32, do not transmit Lucifer yellow to any of the alpha-type connexins analyzed. Among alpha- type connexins, Cx40 does not communicate with Cx43. Thus, connexins differ in their ability to form functional heterotypic gap junctions among mammalian cells.

Full Text

The Full Text of this article is available as a PDF (5.2 MB).

Selected References

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

  1. Barrio L. C., Suchyna T., Bargiello T., Xu L. X., Roginski R. S., Bennett M. V., Nicholson B. J. Gap junctions formed by connexins 26 and 32 alone and in combination are differently affected by applied voltage. Proc Natl Acad Sci U S A. 1991 Oct 1;88(19):8410–8414. doi: 10.1073/pnas.88.19.8410. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. 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]
  3. Bennett M. V., Barrio L. C., Bargiello T. A., Spray D. C., Hertzberg E., Sáez J. C. Gap junctions: new tools, new answers, new questions. Neuron. 1991 Mar;6(3):305–320. doi: 10.1016/0896-6273(91)90241-q. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. Beyer E. C., Reed K. E., Westphale E. M., Kanter H. L., Larson D. M. Molecular cloning and expression of rat connexin40, a gap junction protein expressed in vascular smooth muscle. J Membr Biol. 1992 Apr;127(1):69–76. doi: 10.1007/BF00232759. [DOI] [PubMed] [Google Scholar]
  6. Brissette J. L., Kumar N. M., Gilula N. B., Hall J. E., Dotto G. P. Switch in gap junction protein expression is associated with selective changes in junctional permeability during keratinocyte differentiation. Proc Natl Acad Sci U S A. 1994 Jul 5;91(14):6453–6457. doi: 10.1073/pnas.91.14.6453. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. 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]
  8. Butterweck A., Elfgang C., Willecke K., Traub O. Differential expression of the gap junction proteins connexin45, -43, -40, -31, and -26 in mouse skin. Eur J Cell Biol. 1994 Oct;65(1):152–163. [PubMed] [Google Scholar]
  9. Butterweck A., Gergs U., Elfgang C., Willecke K., Traub O. Immunochemical characterization of the gap junction protein connexin45 in mouse kidney and transfected human HeLa cells. J Membr Biol. 1994 Sep;141(3):247–256. doi: 10.1007/BF00235134. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. 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]
  12. 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]
  13. 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]
  14. Dermietzel R., Spray D. C. Gap junctions in the brain: where, what type, how many and why? Trends Neurosci. 1993 May;16(5):186–192. doi: 10.1016/0166-2236(93)90151-b. [DOI] [PubMed] [Google Scholar]
  15. Dürst M., Dzarlieva-Petrusevska R. T., Boukamp P., Fusenig N. E., Gissmann L. Molecular and cytogenetic analysis of immortalized human primary keratinocytes obtained after transfection with human papillomavirus type 16 DNA. Oncogene. 1987;1(3):251–256. [PubMed] [Google Scholar]
  16. Flagg-Newton J., Simpson I., Loewenstein W. R. Permeability of the cell-to-cell membrane channels in mammalian cell juncton. Science. 1979 Jul 27;205(4404):404–407. doi: 10.1126/science.377490. [DOI] [PubMed] [Google Scholar]
  17. Gho M. Voltage-clamp analysis of gap junctions between embryonic muscles in Drosophila. J Physiol. 1994 Dec 1;481(Pt 2):371–383. doi: 10.1113/jphysiol.1994.sp020446. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Goliger J. A., Paul D. L. Expression of gap junction proteins Cx26, Cx31.1, Cx37, and Cx43 in developing and mature rat epidermis. Dev Dyn. 1994 May;200(1):1–13. doi: 10.1002/aja.1002000102. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Hennemann H., Schwarz H. J., Willecke K. Characterization of gap junction genes expressed in F9 embryonic carcinoma cells: molecular cloning of mouse connexin31 and -45 cDNAs. Eur J Cell Biol. 1992 Feb;57(1):51–58. [PubMed] [Google Scholar]
  21. 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]
  22. Hoh J. H., John S. A., Revel J. P. Molecular cloning and characterization of a new member of the gap junction gene family, connexin-31. J Biol Chem. 1991 Apr 5;266(10):6524–6531. [PubMed] [Google Scholar]
  23. 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]
  24. Imanaga I., Kameyama M., Irisawa H. Cell-to-cell diffusion of fluorescent dyes in paired ventricular cells. Am J Physiol. 1987 Jan;252(1 Pt 2):H223–H232. doi: 10.1152/ajpheart.1987.252.1.H223. [DOI] [PubMed] [Google Scholar]
  25. Jongen W. M., Fitzgerald D. J., Asamoto M., Piccoli C., Slaga T. J., Gros D., Takeichi M., Yamasaki H. Regulation of connexin 43-mediated gap junctional intercellular communication by Ca2+ in mouse epidermal cells is controlled by E-cadherin. J Cell Biol. 1991 Aug;114(3):545–555. doi: 10.1083/jcb.114.3.545. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Kasai H., Petersen O. H. Spatial dynamics of second messengers: IP3 and cAMP as long-range and associative messengers. Trends Neurosci. 1994 Mar;17(3):95–101. doi: 10.1016/0166-2236(94)90112-0. [DOI] [PubMed] [Google Scholar]
  27. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  28. Moreno A. P., Fishman G. I., Spray D. C. Phosphorylation shifts unitary conductance and modifies voltage dependent kinetics of human connexin43 gap junction channels. Biophys J. 1992 Apr;62(1):51–53. doi: 10.1016/S0006-3495(92)81775-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Neyton J., Trautmann A. Single-channel currents of an intercellular junction. 1985 Sep 26-Oct 2Nature. 317(6035):331–335. doi: 10.1038/317331a0. [DOI] [PubMed] [Google Scholar]
  30. Nicholson B., Dermietzel R., Teplow D., Traub O., Willecke K., Revel J. P. Two homologous protein components of hepatic gap junctions. Nature. 1987 Oct 22;329(6141):732–734. doi: 10.1038/329732a0. [DOI] [PubMed] [Google Scholar]
  31. 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]
  32. Pitts J. D., Finbow M. E., Kam E. Junctional communication and cellular differentiation. Br J Cancer Suppl. 1988 Dec;9:52–57. [PMC free article] [PubMed] [Google Scholar]
  33. Quade K. Transformation of mammalian cells by avian myelocytomatosis virus and avian erythroblastosis virus. Virology. 1979 Oct 30;98(2):461–465. doi: 10.1016/0042-6822(79)90569-5. [DOI] [PubMed] [Google Scholar]
  34. Risek B., Guthrie S., Kumar N., Gilula N. B. Modulation of gap junction transcript and protein expression during pregnancy in the rat. J Cell Biol. 1990 Feb;110(2):269–282. doi: 10.1083/jcb.110.2.269. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Salomon D., Saurat J. H., Meda P. Cell-to-cell communication within intact human skin. J Clin Invest. 1988 Jul;82(1):248–254. doi: 10.1172/JCI113578. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Steinberg T. H., Civitelli R., Geist S. T., Robertson A. J., Hick E., Veenstra R. D., Wang H. Z., Warlow P. M., Westphale E. M., Laing J. G. Connexin43 and connexin45 form gap junctions with different molecular permeabilities in osteoblastic cells. EMBO J. 1994 Feb 15;13(4):744–750. doi: 10.1002/j.1460-2075.1994.tb06316.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Stutenkemper R., Geisse S., Schwarz H. J., Look J., Traub O., Nicholson B. J., Willecke K. The hepatocyte-specific phenotype of murine liver cells correlates with high expression of connexin32 and connexin26 but very low expression of connexin43. Exp Cell Res. 1992 Jul;201(1):43–54. doi: 10.1016/0014-4827(92)90346-a. [DOI] [PubMed] [Google Scholar]
  38. Swenson K. I., Jordan J. R., Beyer E. C., Paul D. L. Formation of gap junctions by expression of connexins in Xenopus oocyte pairs. Cell. 1989 Apr 7;57(1):145–155. doi: 10.1016/0092-8674(89)90180-3. [DOI] [PubMed] [Google Scholar]
  39. Tomasetto C., Neveu M. J., Daley J., Horan P. K., Sager R. Specificity of gap junction communication among human mammary cells and connexin transfectants in culture. J Cell Biol. 1993 Jul;122(1):157–167. doi: 10.1083/jcb.122.1.157. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. 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]
  41. Traub O., Look J., Paul D., Willecke K. Cyclic adenosine monophosphate stimulates biosynthesis and phosphorylation of the 26 kDa gap junction protein in cultured mouse hepatocytes. Eur J Cell Biol. 1987 Feb;43(1):48–54. [PubMed] [Google Scholar]
  42. Veenstra R. D., Wang H. Z., Beyer E. C., Brink P. R. Selective dye and ionic permeability of gap junction channels formed by connexin45. Circ Res. 1994 Sep;75(3):483–490. doi: 10.1161/01.res.75.3.483. [DOI] [PubMed] [Google Scholar]
  43. Veenstra R. D., Wang H. Z., Beyer E. C., Ramanan S. V., Brink P. R. Connexin37 forms high conductance gap junction channels with subconductance state activity and selective dye and ionic permeabilities. Biophys J. 1994 Jun;66(6):1915–1928. doi: 10.1016/S0006-3495(94)80985-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Werner R., Levine E., Rabadan-Diehl C., Dahl G. Formation of hybrid cell-cell channels. Proc Natl Acad Sci U S A. 1989 Jul;86(14):5380–5384. doi: 10.1073/pnas.86.14.5380. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. White T. W., Bruzzone R., Goodenough D. A., Paul D. L. Voltage gating of connexins. Nature. 1994 Sep 15;371(6494):208–209. doi: 10.1038/371208a0. [DOI] [PubMed] [Google Scholar]
  46. 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]
  47. Wigler M., Pellicer A., Silverstein S., Axel R. Biochemical transfer of single-copy eucaryotic genes using total cellular DNA as donor. Cell. 1978 Jul;14(3):725–731. doi: 10.1016/0092-8674(78)90254-4. [DOI] [PubMed] [Google Scholar]
  48. Wilders R., Jongsma H. J. Limitations of the dual voltage clamp method in assaying conductance and kinetics of gap junction channels. Biophys J. 1992 Oct;63(4):942–953. doi: 10.1016/S0006-3495(92)81664-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Willecke K., Hennemann H., Dahl E., Jungbluth S., Heynkes R. The diversity of connexin genes encoding gap junctional proteins. Eur J Cell Biol. 1991 Oct;56(1):1–7. [PubMed] [Google Scholar]
  50. 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]
  51. 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]
  52. Zhu D., Caveney S., Kidder G. M., Naus C. C. Transfection of C6 glioma cells with connexin 43 cDNA: analysis of expression, intercellular coupling, and cell proliferation. Proc Natl Acad Sci U S A. 1991 Mar 1;88(5):1883–1887. doi: 10.1073/pnas.88.5.1883. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES